COMPOSITIONS OF MATTER FOR DETECTION ASSAYS

Abstract

The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5′-to-3′ direction comprises: A-(B-L)J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

Description

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 17/861,208, filed 9 Jul. 2022, which claims priority to U.S. Ser. No. 63/220,987, filed 12 Jul. 2021, and U.S. Ser. No. 63/289,112, filed 13 Dec. 2021.

FIELD OF THE INVENTION

The present disclosure relates to methods, compositions of matter and assay systems used to detect one or more target nucleic acids of interest in a sample. The assay systems provide signal amplification upon detection of a target nucleic acids of interest without amplification of the target nucleic acids.

INCORPORATION BY REFERENCE

Submitted on 4 Aug. 2022 is an electronically filed sequence listing via EFS-Web a Sequence Listing XML, entitled “LS002US2_seqlist_20220804”, created 4 Aug. 2022, which is 6,799,000 bytes in size. The sequence listing is part of the specification filed 9 Jul. 2022 and is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

Rapid and accurate identification of infectious agents is important in order to select correct treatment and prevent further spreading of viral infections and pandemic diseases. For example, viral pathogens, such as SARS-CoV-2, and the associated COVID-19 disease require immediate detection and response to decrease mortality, morbidity and transmission.

Classic nucleic acid-guided nuclease or CRISPR (clustered regularly interspaced short palindromic repeats) detection methods usually rely on pre-amplification of target nucleic acids of interest to enhance detection sensitivity. However, amplification increases time to detection and may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results. Improved technologies that allow very rapid and accurate detection of pathogens are therefore needed for timely diagnosis, prevention and treatment of disease, as well as in other applications.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims. Further, all of the functionalities described in connection with one embodiment of the compositions and methods described herein are intended to be applicable to the additional embodiments of the compositions and methods described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

The present disclosure provides compositions of matter, methods, and cascade assays to detect target nucleic acids of interest. The cascade assays described herein comprise two different ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep one of the ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the other ribonucleoprotein complex. The present nucleic acid-guided nuclease cascade assay can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected. In this sense, the cascade assay is modular.

There is provided herein in one embodiment of the disclosure a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules cannot activate the RNP1 or the RNP2.

There is provided in a second embodiment of the disclosure, a reaction mixture comprising: (i) a first complex comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecule cannot activate the first or second complex.

Provided in a third embodiment is a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) (RNP1) complex comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both sequence-specific activity and non-sequence-specific activity; (ii) a second ribonucleoprotein (RNP2) complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both sequence-specific activity and non-sequence-specific activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules do not bind to the RNP1 complex or the RNP2 complex. In yet another fourth embodiment of the disclosure there is provided a reaction mixture comprising: (i) a first complex comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both sequence-specific activity and non-sequence-specific activity; (ii) a second complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both sequence-specific activity and non-sequence-specific activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules are not recognized by the RNP1s or RNP2s.

A fifth embodiment provides a cascade assay method for detecting a target nucleic acid of interest in a sample comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules cannot activate the RNP1 or the RNP2; (b) contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1; wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule and the at least one unblocked nucleic acid molecule binds to RNP2 initiating cleavage of at least one further blocked nucleic acid molecule; and (c) detecting products of the cleavage, thereby detecting the target nucleic acid of interest in the sample.

In a sixth embodiment there is provided a kit for detecting a target nucleic acid of interest in a sample comprising: (i) a first ribonucleoprotein (RNP1) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; a (iii) plurality of blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein trans-cleavage activity of the blocked nucleic acid molecules results in at least one unblocked nucleic acid molecule; and wherein the unblocked nucleic acid molecule activates trans-cleavage activity of the second nucleic acid-guided nuclease in at least one RNP2 initiating cleavage of more blocked nucleic acid molecules; and (iv) a reporter moiety, wherein the reporter molecule comprises a nucleic acid molecule and/or is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon trans-cleavage activity by the RNP1 or the RNP2, to identify the presence of the target nucleic acid of interest in the sample.

In some aspects of any one of the aforementioned embodiments, the first and/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease can is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects, the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects of any one of the aforementioned embodiments, the blocked nucleic acid molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV comprise in the 5′-to-3′ direction:

(a)

A-(B-L)_J-C-M-T-D  (Formula I);

• • wherein A is 0-15 nucleotides in length;
  • B is 4-12 nucleotides in length;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10;
  • C is 4-15 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)_J-C and T-D are separate nucleic acid strands;
  • T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and
  • D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A;

(b)

D-T-T′-C-(L-B)_J-A  (Formula II);

• • wherein D is 0-10 nucleotides in length;
  • T-T′ is 17-135 nucleotides in length;
  • T′ is 1-10 nucleotides in length and does not hybridize with T;
  • C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length and does not hybridize with T;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • J is an integer between 1 and 10;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;

(c)

T-D-M-A-(B-L)_J-C  (Formula III);

• • wherein T is 17-135 nucleotides in length;
  • D is 0-10 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)_J-C are separate nucleic acid strands;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10; and
  • C is 4-15 nucleotides in length; or

(d)

T-D-M-A-L_p-C  (Formula IV);

• • wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
  • D is 0-15 nucleotides in length;
  • M is 1-25 nucleotides in length;
  • A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
  • L is 3-25 nucleotides in length;
  • p is 0 or 1;
  • C is 4-15 nucleotides in length and comprises a sequence complementary to T.
    And in some aspects,
  • (a) T of Formula I comprises at least 80% sequence complementarity to B and C;
  • (b) D of Formula I comprises at least 80% sequence complementarity to A;
  • (c) C of Formula II comprises at least 80% sequence complementarity to T;
  • (d) B of Formula II comprises at least 80% sequence complementarity to T;
  • (e) A of Formula II comprises at least 80% sequence complementarity to D;
  • (f) A of Formula III comprises at least 80% sequence complementarity to D;
  • (g) B of Formular III comprises at least 80% sequence complementarity to T;
  • (h) A of Formula IV comprises at least 80% sequence complementarity to D; and/or
  • (i) C of Formula IV comprises at least 80% sequence complementarity to T.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules comprise a first sequence complementary to the second gRNA and a second sequence not complementary to the second gRNA, wherein the second sequence at least partially hybridizes to the first sequence resulting in at least one loop.

In some aspects of the aforementioned embodiments, the reaction mixture comprises about 1 fM to about 10 μM of the RNP1 and in some aspects the reaction mixture comprises about 1 fM to about 1 mM of the RNP2.

In some aspects of the aforementioned embodiments, the reaction mixture comprises at least two different RNP1s, wherein different RNP1s comprise different gRNA sequences, and in some aspects the reaction mixture comprises 2 to 10000 different RNP1s, or 2 to 1000 different RNP1s, or 2 to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules include the sequence of any one of SEQ ID NOs: 14-1421.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules are circular, and in some aspects the blocked nucleic acid molecules are linear.

In some aspects the K_d of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_d of unblocked nucleic acid molecules.

In some aspects of the aforementioned embodiments, the target nucleic acid of interest is of bacterial, viral, fungal, mammalian or plant origin, and in some aspects, the sample may include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood; food; agricultural products; pharmaceuticals; cosmetics, nutriceuticals; personal care products; environmental substances such as soil, water, or air; industrial sites and products; or manufactured or natural chemicals and compounds.

In some aspects of the aforementioned embodiments, the reaction mixture further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules do not comprise a PAM sequence, yet in other aspects, the blocked nucleic acid molecules comprise a PAM sequence, and in some aspects the PAM sequence is disposed between the first and second sequences, wherein the first sequence is 5′ to the PAM sequence.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecule is a blocked primer molecule.

In a seventh embodiment of the disclosure, there is provided a blocked nucleic acid molecule comprising: a first region recognized by a ribonucleoprotein (RNP) complex; one or more second regions not complementary to the first region; and one or more third regions complementary and hybridized to the first region, wherein cleavage of the one or more second regions results in dehybridization of the third region from the first region, resulting in an unblocked nucleic acid molecule.

An eighth embodiment provides a method of unblocking a blocked nucleic acid comprising: (a) providing a blocked nucleic acid molecule comprising: a first region recognized by a ribonucleoprotein (RNP) complex; one or more second regions not complementary to the first region; and one or more third regions complementary and hybridized to the first region, wherein cleavage of the one or more second regions results in dehybridization of the third region from the first region, resulting in an unblocked nucleic acid molecule; and (b) initiating cleavage of the one or more second regions, wherein the blocked nucleic acid molecule becomes an unblocked nucleic acid molecule.

A ninth embodiment provides a composition of matter comprising: a first region recognized by a ribonucleoprotein (RNP) complex; one or more second regions of not complementary to the first region; and one or more third regions complementary and hybridized to the first region, wherein cleavage of the one or more second regions results in dehybridization of the third region from the first region, resulting in an unblocked nucleic acid molecule; and the RNP complex comprising a gRNA that is complementary to the first region and a nucleic acid-guided nuclease, wherein the nucleic acid-guided nuclease exhibits both sequence-specific and non-sequence-specific nuclease activity.

Additionally, a tenth embodiment of the disclosure provides a cascade assay method of detecting a target nucleic acid of interest in a sample comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and (iii) a plurality of linear blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the linear blocked nucleic acid molecules cannot activate the RNP1 or the RNP2; (b) contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1; wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the linear blocked nucleic acid molecules thereby producing at least one linear unblocked nucleic acid molecule and the at least one linear unblocked nucleic acid molecule to RNP2 initiating cleavage of at least one further linear blocked nucleic acid molecule; and (c) detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

In some aspects of any one of the aforementioned embodiments, the first and/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease can is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects, the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects, the blocked nucleic acid molecule comprises a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction:

(a)

A-(B-L)_J-C-M-T-D  (Formula I);

• • wherein A is 0-15 nucleotides in length;
  • B is 4-12 nucleotides in length;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10;
  • C is 4-15 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)_J-C and T-D are separate nucleic acid strands;
  • T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and
  • D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A;

(b)

D-T-T′-C-(L-B)_J-A  (Formula II);

• • wherein D is 0-10 nucleotides in length;
  • T-T′ is 17-135 nucleotides in length;
  • T′ is 1-10 nucleotides in length and does not hybridize with T;
  • C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length and does not hybridize with T;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • J is an integer between 1 and 10;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;

(c)

T-D-M-A-(B-L)_J-C  (Formula III);

• • wherein T is 17-135 nucleotides in length;
  • D is 0-10 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)_J-C are separate nucleic acid strands;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10; and
  • C is 4-15 nucleotides in length; or

(d)

T-D-M-A-L_p-C  (Formula IV);

• • wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
  • D is 0-15 nucleotides in length;
  • M is 1-25 nucleotides in length;
  • A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
  • L is 3-25 nucleotides in length;
  • p is 0 or 1;
  • C is 4-15 nucleotides in length and comprises a sequence complementary to T.

Further

• • (a) T of Formula I comprises at least 80% sequence complementarity to B and C;
  • (b) D of Formula I comprises at least 80% sequence complementarity to A;
  • (c) C of Formula II comprises at least 80% sequence complementarity to T;
  • (d) B of Formula II comprises at least 80% sequence complementarity to T;
  • (e) A of Formula II comprises at least 80% sequence complementarity to D;
  • (f) A of Formula III comprises at least 80% sequence complementarity to D;
  • (g) B of Formular III comprises at least 80% sequence complementarity to T;
  • (h) A of Formula IV comprises at least 80% sequence complementarity to D; and/or
  • (i) C of Formula IV comprises at least 80% sequence complementarity to T.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecule comprises a modified nucleoside or nucleotide, including but not limited to a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond. In some aspects, the blocked nucleic acid molecule includes the sequence of any one of SEQ ID NOs: 14-1421; the blocked nucleic acid molecule is a blocked primer molecule; the blocked nucleic acid molecule does not comprise a PAM sequence; and/or in some aspects the blocked nucleic acid molecule comprises a PAM sequence, and the PAM sequence is disposed between the first and second sequences, wherein the first sequence is 5′ to the PAM sequence.

In some aspects of the aforementioned embodiments, the reaction mixture further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects, the K_d of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_d of unblocked nucleic acid molecules.

In an eleventh embodiment, there is provided composition of matter comprising a ribonucleoprotein (RNP) complex and a blocked nucleic acid molecule, wherein the blocked nucleic acid molecule is represented by any one of Formula I-IV, wherein Formulas I— IV comprise in the 5′-to-3′ direction comprises:

(a)

A-(B-L)_J-C-M-T-D  (Formula I);

• • wherein A is 0-15 nucleotides in length;
  • B is 4-12 nucleotides in length;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10;
  • C is 4-15 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)_J-C and T-D are separate nucleic acid strands;
  • T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and
  • D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A;

(b)

D-T-T′-C-(L-B)_J-A  (Formula II);

• • wherein D is 0-10 nucleotides in length;
  • T-T′ is 17-135 nucleotides in length;
  • T′ is 1-10 nucleotides in length and does not hybridize with T;
  • C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length and does not hybridize with T;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • J is an integer between 1 and 10;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;

(c)

T-D-M-A-(B-L)_J-C  (Formula III);

• • wherein T is 17-135 nucleotides in length;
  • D is 0-10 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)_J-C are separate nucleic acid strands;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10; and
  • C is 4-15 nucleotides in length; or

(d)

T-D-M-A-L_p-C  (Formula IV);

• • wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
  • D is 0-15 nucleotides in length;
  • M is 1-25 nucleotides in length;
  • A is 0-15 nucleotides in length and comprises a sequence complementary to D;
    • and
  • L is 3-25 nucleotides in length;
  • p is 0 or 1;
  • C is 4-15 nucleotides in length and comprises a sequence complementary to T.

In some aspects of this embodiment,

T of Formula I comprises at least 80% sequence complementarity to B and C;

• • (a) D of Formula I comprises at least 80% sequence complementarity to A;
  • (b) C of Formula II comprises at least 80% sequence complementarity to T;
  • (c) B of Formula II comprises at least 80% sequence complementarity to T;
  • (d) A of Formula II comprises at least 80% sequence complementarity to D;
  • (e) A of Formula III comprises at least 80% sequence complementarity to D;
  • (f) B of Formular III comprises at least 80% sequence complementarity to T;
  • (g) A of Formula IV comprises at least 80% sequence complementarity to D; and/or
  • (h) C of Formula IV comprises at least 80% sequence complementarity to T.

In some aspects of the aforementioned embodiment, the blocked primer molecules include the sequence of any one of SEQ ID NOs: 14-1421.

In some aspects of the aforementioned embodiment, the RNP complex comprises a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the RNP complex comprises a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the RNP complex comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects of the aforementioned embodiment, the blocked nucleic acid molecule comprises a modified nucleoside or nucleotide comprises a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.

In some aspects, the blocked nucleic acid molecule does not comprise a PAM sequence, and in other aspects, the blocked nucleic acid molecule comprises a PAM sequence where the PAM sequence is disposed between the first and second sequences, wherein the first sequence is 5′ to the PAM sequence. In some aspects, the blocked nucleic acid molecule is a blocked primer molecule.

In some aspects of the aforementioned embodiment(s), the composition of matter further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

Yet another embodiment provides a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (iii) a plurality of template molecules comprising a sequence corresponding to the second gRNA; (iv) a plurality of blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked nucleic acid molecules cannot be extended by a polymerase; and (v) a polymerase and a plurality of nucleotides.

Another embodiment provides a cascade assay method for detecting a target nucleic acid of interest in a sample comprising: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (iii) a plurality of template molecules comprising a sequence corresponding to the second gRNA; (iv) a plurality of blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked nucleic acid molecules cannot be extended by a polymerase; and (v) a polymerase and a plurality of nucleotides; (b) contacting the reaction mixture with the sample under conditions that allow target nucleic acids of interest in the sample to bind to the first gRNA, wherein: upon binding of the target nucleic acid of interest, the RNP1 active cleaving at least one of the blocked primer molecules, thereby producing at least one unblocked primer molecule that can be extended by the polymerase; at least one unblocked primer molecule binds to one of the template molecules and is extended by the polymerase and nucleotides to form at least one synthesized activating molecule having a sequence complementary to the second gRNA; at least one synthesized activating molecule binds to the second gRNA, and RNP2 becomes active cleaving at least one further blocked primer molecule; and detecting the cleavage products of step (b), thereby detecting the target nucleic acid of interest in the sample.

In some aspects the K_d of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_d of unblocked nucleic acid molecules.

A further embodiment provides a kit for detecting a target nucleic acid of interest in a sample comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; (iii) a plurality of template molecules comprising a non-target sequence to the second gRNA; (iv) a polymerase and nucleotides; (v) a plurality of blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecule cannot be extended by the polymerase, trans-cleavage of the blocked primer molecules results in at least one unblocked primer molecule; and wherein the unblocked primer molecule can bind to at least one the template molecule and be extended by the polymerase to form a synthesized activating molecule; and (vi) a reporter moiety, wherein the reporter moiety comprises a nucleic acid molecule and/or is operably linked to the blocked primer molecule and produces a detectable signal upon trans-cleavage activity of the blocked primer molecule by the RNP1 or the RNP2, to identify the presence of the target nucleic acid of interest in the sample.

In any of these embodiments, the first and/or second nucleic acid-guided nuclease in the reaction mixture is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease; and in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects the blocked primer molecules comprise a first sequence complementary to the second gRNA and a second sequence not complementary to the second gRNA, wherein the second sequence at least partially hybridizes to the first sequence resulting in at least one loop; and in some aspects, the blocked primer molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction:

(a)

A-(B-L)_J-C-M-T-D  (Formula I);

• • wherein A is 0-15 nucleotides in length;
  • B is 4-12 nucleotides in length;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10;
  • C is 4-15 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)_J-C and T-D are separate nucleic acid strands;
  • T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and
  • D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A;

(b)

D-T-T′-C-(L-B)_J-A  (Formula II);

• • wherein D is 0-10 nucleotides in length;
  • T-T′ is 17-135 nucleotides in length;
  • T′ is 1-10 nucleotides in length and does not hybridize with T;
  • C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length and does not hybridize with T;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • J is an integer between 1 and 10;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;

(c)

T-D-M-A-(B-L)_J-C  (Formula III);

• • wherein T is 17-135 nucleotides in length;
  • D is 0-10 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)_J-C are separate nucleic acid strands;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10; and
  • C is 4-15 nucleotides in length; or

(d)

T-D-M-A-L_p-C  (Formula IV);

• • wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
  • D is 0-15 nucleotides in length;
  • M is 1-25 nucleotides in length;
  • A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
  • L is 3-25 nucleotides in length;
  • p is 0 or 1;
  • C is 4-15 nucleotides in length and comprises a sequence complementary to T.
    In some aspects,
  • (a) T of Formula I comprises at least 80% sequence complementarity to B and C;
  • (b) D of Formula I comprises at least 80% sequence complementarity to A;
  • (c) C of Formula II comprises at least 80% sequence complementarity to T;
  • (d) B of Formula II comprises at least 80% sequence complementarity to T;
  • (e) A of Formula II comprises at least 80% sequence complementarity to D;
  • (f) A of Formula III comprises at least 80% sequence complementarity to D;
  • (g) B of Formular III comprises at least 80% sequence complementarity to T;
  • (h) A of Formula IV comprises at least 80% sequence complementarity to D; and/or
  • (i) C of Formula IV comprises at least 80% sequence complementarity to T.

In some aspects the reaction mixture comprises about 1 fM to about 10 μM of the RNP1, and in some aspects, the reaction mixture of claim 1, wherein the reaction mixture comprises about 1 fM to about 1 mM of the RNP2.

In some aspects of these embodiments, the reaction mixture comprises at least two different RNP 1 s, wherein different RNP 1 s comprise different gRNA sequences, and in some aspects, the reaction mixture comprises 2 to 10000 different RNP1s, 2 to 1000 different RNP1s, 2 to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects the blocked primer molecules include the sequence of any one of SEQ ID NOs: 14-1421. In some aspects the K_d of the blocked primer molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_d of unblocked primer molecules.

In some aspects of the aforementioned embodiments, the reaction mixture further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects of the aforementioned embodiments, the template molecules do not comprise a complement of a PAM sequence, and in some aspects, the template molecules comprise a complement of a PAM sequence. In some aspects, the template molecules are single-stranded. In some aspects, the template molecules are linear; in yet other aspects the template molecules are circularized. In some aspects comprising circular blocked nucleic acid molecules, at least one of the plurality of circular high Kd blocked nucleic acid molecules comprises a first region comprising a sequence specific to the second guide RNA and a second region comprising a nuclease-cleavable sequence; where at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA sequence in the second region; at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; or at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable RNA sequence in the second region.

In some aspects the polymerase comprises strand displacement activity and/or 3′-to-5′ exonuclease activity, and in some aspects, the polymerase is Phi29 polymerase.

Yet another embodiment provides a composition of matter comprising a circular high Kd blocked nucleic acid molecule comprising: a region recognized by a gRNA in an RNP complex; a region comprising a sequence cleavable by a nucleic acid-guided nuclease in the RNP complex; and wherein the circular high Kd blocked nucleic acid molecule cannot activate the RNP complex, and wherein the circular high Kd blocked nucleic molecules are high Kd in relation to binding to the RNP complex.

A further embodiment provides a method of unblocking a circular high K_d blocked nucleic acid molecule comprising the steps of: (a) providing a circular high K_d blocked nucleic acid molecule comprising: a first region recognized by a gRNA in an RNP complex; a second region comprising a sequence cleavable by a nucleic acid-guided nuclease in the RNP complex, wherein the circular high K_d blocked nucleic acid molecule cannot substantially activate the RNP complex; and (b) initiating cleavage of the second region by the nucleic acid-guided nuclease in the RNP complex, wherein the circular high K_d blocked nucleic acid molecule becomes a linear low K_d unblocked nucleic acid molecule, and wherein the circular high K_d blocked nucleic acid molecules are high K_d and linear low K_d unblocked nucleic acid molecules are high K_d and low K_d in relation to binding the RNP complex.

Also provided as an embodiment is a cascade assay method of detecting a target nucleic acid of interest in a sample comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest; (ii) a second ribonucleoprotein (RNP2) complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of circular blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the circular blocked nucleic acid molecules cannot activate the RNP1 complex or the RNP2 complex; (b) contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1; wherein upon binding of the target nucleic acid of interest, RNP1 becomes active initiating trans-cleavage of at least one of the circular blocked nucleic acid molecules thereby producing at least one linear unblocked nucleic acid molecule; the at least one linear unblocked nucleic acid molecule binds to RNP2 initiating cleavage of at least one further circular blocked nucleic acid molecule; and (c) detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

In some aspects, the RNP complex (either RNP1 or RNP2) comprises a nucleic acid-guided nuclease selected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b, and in some aspects, the RNP complex comprises a nucleic acid-guided nuclease that is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease; the RNP complex comprises a nucleic acid-guided nuclease that exhibits both cis-cleavage and trans-cleavage activity; and/or the RNP complex comprises a nucleic acid-guided nuclease comprising a RuvC nuclease domain or a RuvC-like nuclease domain but lacks an HNH nuclease domain.

In some aspects of any embodiments comprising circular high K_d blocked nucleic acid molecules, the circular high K_d blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA sequence in the second region; the circular high K_d blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; the circular high K_d blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; or the circular high K_d blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable RNA sequence in the second region.

In some aspects of these embodiments, the circular high K_d blocked nucleic acid molecule comprises 5′ and 3′ ends hybridized to each other and DNA, RNA, LNA or PNA bases having a high T_m; and in some aspects, the K_d of the circular high K_d blocked nucleic acid molecules to the RNP complex or RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_d of unblocked circular high K_d blocked nucleic acid molecules.

In some aspects the circular high K_d blocked nucleic acid molecule comprises a modified nucleoside or nucleotide, including but not limited to a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.

In some aspects the circular high K_d blocked nucleic acid molecule is a single-stranded, double-stranded, or partially double-stranded molecule comprising one or more different combinations of DNA-DNA, DNA-RNA or RNA-RNA hybrid molecules. In some aspects the circular high K_d blocked nucleic acid molecule is a circular high K_d primer molecule. In some aspects the circular high K_d blocked nucleic acid molecule does not comprise a PAM sequence or the circular high K_d blocked nucleic acid molecule comprises a PAM sequence.

In some aspects of the aforementioned embodiments, the compositions of matter or reaction further comprises a reporter moiety wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

Yet another embodiment provides a composition of matter comprising: (a) a first preassembled ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA that is specific to a target nucleic acid of interest, wherein the first nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; (b) a second preassembled ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second guide RNA, wherein the second nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; and (c) a plurality of circular high K_d blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the circular high K_d blocked nucleic acid molecules are not recognized by the RNP1 or RNP2, and wherein the circular high K_d blocked nucleic acid molecules are high K_d in relation to binding to RNP2.

Another embodiment provides a composition of matter comprising: (a) a first preassembled ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA that is specific to a target nucleic acid of interest, wherein the first nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; (b) a second preassembled ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second guide RNA, wherein the second nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; and (c) a plurality of engineered linear high K_d blocked nucleic acid molecules comprising a first sequence complementary to the second gRNA, wherein the linear high K_d blocked nucleic acid molecules are not recognized by the RNP1 and RNP2, and wherein the linear high K_d blocked nucleic acid molecules are high K_d in relation to binding to the RNP2.

Yet another embodiment provides a composition of matter comprising: (a) a first preassembled ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA that is specific to a target nucleic acid of interest, wherein the first nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; (b) a second preassembled ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second guide RNA, wherein the second nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; and (c) a plurality of engineered high K_d blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the high K_d blocked nucleic acid molecules are not recognized by the RNP1 and RNP2, and wherein the high K_d blocked nucleic acid molecules are high K_d in relation to binding to the RNP complex.

In aspects of any one of the foregoing embodiments, the high K_d blocked nucleic acid molecule comprises DNA, RNA, LNA or PNA bases having a high Tm; the 5′ and 3′ ends of the high K_d blocked nucleic acid molecule comprise phosphorothioate bonds (PS); high K_d blocked nucleic acid molecule comprises one or more different combinations of DNA-DNA, DNA-RNA or RNA-RNA hybrid molecules; and/or the high K_d blocked nucleic acid molecule comprises a nucleic acid region comprising nanoparticles attached thereto, wherein the nanoparticles provide steric hindrance to internalization in RNP2 and block RNP2 activation until cleavage and removal of the nucleic acid region comprising the nanoparticles.

In other aspects, the first and/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease can is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects, the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

Aspects also include the composition of matter comprises about 1 fM to about 10 μM of the RNP1; and/or the composition of matter comprises about 1 fM to about 1 mM of the RNP2.

In some aspects the composition of matter comprises at least two different RNP1 complex species, wherein different RNP 1s comprise different gRNA sequences; and in some aspects the composition comprises 2 to 10000 different RNP1s, 2 to 1000 different RNP1s, 2 to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects the RNP2 recognizes a PAM sequence, and in other aspects the RNP2 complex does not recognize a PAM sequence.

In some aspects of the aforementioned embodiments, the composition of matter further comprises a reporter moiety, wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects the high Kd blocked nucleic acid molecule is a high Kd blocked primer molecule.

In some aspects the linear high K_d blocked nucleic acid molecule is converted to a linear low K_d blocked nucleic acid molecule upon trans-cleavage by RNP1 and/or RNP2. In some aspects the K_d of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_d of unblocked nucleic acid molecules.

In some aspects of the compositions of matter comprising circular blocked nucleic acid molecules, at least one of the plurality of circular high Kd blocked nucleic acid molecules comprises a first region comprising a sequence specific to the second guide RNA and a second region comprising a nuclease-cleavable sequence; where at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA sequence in the second region; at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; or at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable RNA sequence in the second region.

In some aspects of the compositions of matter comprising linear blocked nucleic acid molecules, the linear high K_d nucleic acid molecules comprise a structure represented by any one of Formulas I-IV, where Formulas I-IV comprise in the 5′-to-3′ direction:

(a)

A-(B-L)_J-C-M-T-D  (Formula I);

• • wherein A is 0-15 nucleotides in length;
  • B is 4-12 nucleotides in length;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10;
  • C is 4-15 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)_J-C and T-D are separate nucleic acid strands;
  • T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and
  • D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A;

(b)

D-T-T′-C-(L-B)_J-A  (Formula II);

• • wherein D is 0-10 nucleotides in length;
  • T-T′ is 17-135 nucleotides in length;
  • T′ is 1-10 nucleotides in length and does not hybridize with T;
  • C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length and does not hybridize with T;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • J is an integer between 1 and 10;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;

(c)

T-D-M-A-(B-L)_J-C  (Formula III);

• • wherein T is 17-135 nucleotides in length;
  • D is 0-10 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)_J-C are separate nucleic acid strands;
  • A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;
  • B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10; and
  • C is 4-15 nucleotides in length; or

(d)

T-D-M-A-L_p-C  (Formula IV);

• • wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
  • D is 0-15 nucleotides in length;
  • M is 1-25 nucleotides in length;
  • A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
  • L is 3-25 nucleotides in length;
  • p is 0 or 1;
  • C is 4-15 nucleotides in length and comprises a sequence complementary to T.
    And in some aspects,
  • (a) T of Formula I comprises at least 80% sequence complementarity to B and C;
  • (b) D of Formula I comprises at least 80% sequence complementarity to A;
  • (c) C of Formula II comprises at least 80% sequence complementarity to T;
  • (d) B of Formula II comprises at least 80% sequence complementarity to T;
  • (e) A of Formula II comprises at least 80% sequence complementarity to D;
  • (f) A of Formula III comprises at least 80% sequence complementarity to D;
  • (g) B of Formular III comprises at least 80% sequence complementarity to T;
  • (h) A of Formula IV comprises at least 80% sequence complementarity to D; and/or
  • (i) C of Formula IV comprises at least 80% sequence complementarity to T.

In some aspects, at least one of the linear blocked nucleic acid molecules include the sequence of any one of SEQ ID NOs: 14-1421.

In another embodiment, there is provided a method for syndromic testing comprising: (a) providing a reaction mixture comprising: (i) a plurality of first ribonucleoprotein complexes (RNP1s), each RNP1 comprising a nucleic acid-guided nuclease exhibiting both cis-cleavage and trans-cleavage activity and a first guide RNA (gRNA), wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the reaction mixture comprises at least two different RNP1s, wherein different RNP1s comprise different first gRNAs; (ii) a second ribonucleoprotein complex (RNP2), wherein the RNP2 comprises a second nucleic acid-guided nuclease and a second gRNA that does not recognize any of the target nucleic acids of interest; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecule cannot substantially activate the plurality of RNP1s or the RNP2; (b) contacting the reaction mixture with a sample under conditions that allow target nucleic acids of interest in the sample to bind to the RNP1s, wherein: upon binding of any one of the target nucleic acids of interest, the RNP1 becomes active, cleaving at least one of the blocked nucleic acid molecules, thereby producing at least one unblocked nucleic acid molecule; and at least one unblocked nucleic acid molecule binds to the second gRNA thereby activating RNP2 and initiating trans-cleavage of at least one further blocked nucleic acid molecule; and (c) detecting products of the cleavage of step (b), thus identifying at least one target nucleic acid of interest in the sample.

In some aspects of this embodiment, the first and/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease can is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects, the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

Aspects also include the reaction mixture comprises about 1 fM to about 10 μM of the RNP1; and/or the reaction mixture comprises about 1 fM to about 1 mM of the RNP2. In some aspects the reaction mixture comprises at least two different RNP1 complex species, wherein different RNP1s comprise different gRNA sequences; and in some aspects the composition comprises 2 to 10000 different RNP1s, 2 to 1000 different RNP1s, 2 to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects the K_d of the plurality of blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_d of unblocked nucleic acid molecules.

In some aspects of the aforementioned embodiment, the target nucleic acid of interest is of bacterial, viral, fungal, or mammalian origin, and in some aspects, the sample may include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and/or umbilical cord blood.

In some aspects of the aforementioned embodiments, the reaction mixture further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds. In some aspects the detectable signal is produced within about 1-10 minutes upon the target nucleic acid of interest activating RNP1.

In some aspects the blocked nucleic acid molecules comprise a PAM sequence and in other aspects, the blocked nucleic acid molecules do not comprise a PAM sequence. In some aspects the blocked nucleic acid molecules are linear and in some aspects, the blocked nucleic acids are circular and in yet other aspects, the blocked nucleic acid molecules are a mixture of circular and linear blocked nucleic acid molecules.

In some aspects the blocked nucleic acid molecules are blocked primer molecules and wherein the reaction mixture further comprises a polymerase and nucleotides.

In some aspects, the syndromic testing is for any two or more of common flu (e.g., influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, and influenza B); one of the multiple strains of respiratory syncytial virus (RSV), such as RSV-A and RSV-B; at least one variant of SARS-CoV-2 (e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5); and at least one of other pathogens of interest (e.g., parainfluenza virus 1-4, human metapneumovirus, human rhinovirus, human enterovirus, adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, MERS).

Yet other embodiments provide: a method of detecting a target nucleic acid of interest in a sample comprising the steps of: providing a reaction mixture comprising a first RNP complex comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA), wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest; and a second RNP complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; and contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to the first gRNA, wherein upon binding of the target nucleic acid of interest, the first RNP complex becomes active which catalyzes activation of the second RNP complex via one or more blocked nucleic acids to produce a detectable signal from a reporter moiety.

A further embodiment provides a modular cascade assay comprising: a first nucleic acid-guided nuclease, wherein the first nucleic acid-guided nuclease will form a first ribonucleoprotein complex with a first gRNA that is complementary to a target nucleic acid of interest; a second RNP2 complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to a target nucleic acid of interest; and a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules cannot activate the RNP1 complex or the RNP2 complex; wherein by changing the sequence of the first gRNA, the modular cascade assay is changed to detect different target nucleic acids of interest.

These aspects and other features and advantages of the invention are described below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A is an overview of a prior art assay where target nucleic acids of interest from a sample must be amplified before performing a detection assay.

FIG. 1B is an overview of the general principles underlying the nucleic acid-guided nuclease cascade assay described in detail herein where target nucleic acids of interest from a sample do not need to be amplified before detection.

FIG. 2A is a diagram showing the sequence of steps in an exemplary cascade assay utilizing blocked nucleic acids.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule and a method for unblocking the blocked nucleic acid molecules of the disclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula IV, as described herein.

FIG. 2G shows an exemplary single-stranded blocked nucleic acid molecule with a design able to block R-loop formation with an RNP complex, thereby blocking activation of the trans-nuclease activity of an RNP complex (i.e., RNP2).

FIG. 2H shows schematics of exemplary circularized blocked nucleic acid molecules.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and circular template molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5A shows a lateral flow assay that can be used to detect the cleavage and separation of a signal from a reporter moiety.

FIG. 5B shows a schematic of a lateral flow assay device illustrating the results of an exemplary syndromic test.

FIG. 6 shows a titered quantification of a synthesized nucleocapsid gene (N-gene) using the nucleic acid detection methods described herein. As described in Example VI, a cascade assay was initiated using the detection methods described in Examples II-V above.

FIG. 7 shows titered quantification of an inactivated SARS-CoV-2 virus using the nucleic acid detection methods described in Examples II-V above.

FIG. 8 shows titered quantification of DNA from Methicillin-resistant Staphylococcus (MRSA) using the nucleic acid detection methods described in Examples II-V.

FIG. 9 shows titered quantification of DNA from Methicillin-resistant Staphylococcus (MRSA) using the nucleic acid detection methods described in Examples II-V.

FIG. 10 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C5 as the blocked nucleic acid molecule.

FIG. 11 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C6 as the blocked nucleic acid molecule.

FIG. 12 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C7 as the blocked nucleic acid molecule.

FIG. 13 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C8 as the blocked nucleic acid molecule.

FIG. 14 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C9 as the blocked nucleic acid molecule.

It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.

Definitions

All of the functionalities described in connection with one embodiment of the compositions and methods described herein are intended to be applicable to the additional embodiments of the compositions and methods described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “a system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention. Conventional methods are used for the procedures described herein, such as those provided in the art, and demonstrated in the Examples and various general references. Unless otherwise stated, nucleic acid sequences described herein are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA, as RNA, or a combination of DNA and RNA (e.g., a chimeric nucleic acid).

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

The term “and/or” where used herein is to be taken as specific disclosure of each of the multiple specified features or components with or without another. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

As used herein, the term “about,” as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

As used herein, the terms “binding affinity” or “dissociation constant” or “K_d” refer to the tendency of a molecule to bind (covalently or non-covalently) to a different molecule. A high K_d (which in the context of the present disclosure refers to blocked nucleic acid molecules or blocked primer molecules binding to RNP2) indicates the presence of more unbound molecules, and a low K_d (which in the context of the present disclosure refers to unblocked nucleic acid molecules or unblocked primer molecules binding to RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, aow K_d values are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_d values are in the range of 100 nM-100 μM (10 mM) and thus are about 10⁵- to 10¹⁰-fold or higher as compared to low K_d values.

As used herein, the terms “binding domain” or “binding site” refer to a region on a protein, DNA, or RNA, to which specific molecules and/or ions (ligands) may form a covalent or non-covalent bond. By way of example, a polynucleotide sequence present on a nucleic acid molecule (e.g., a primer binding domain) may serve as a binding domain for a different nucleic acid molecule (e.g., an unblocked primer nucleic acid molecule). Characteristics of binding sites are chemical specificity, a measure of the types of ligands that will bond, and affinity, which is a measure of the strength of the chemical bond.

As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that cannot bind to the first or second RNP complex to activate cis- or trans-cleavage. “Unblocked nucleic acid molecule” refers to a formerly blocked nucleic acid molecule that can bind to the second RNP complex (RNP2) to activate trans-cleavage of additional blocked nucleic acid molecules.

The terms “Cas RNA-guided endonuclease” or “CRISPR nuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associated protein that is an RNA-guided endonuclease suitable for assembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP) complex.

As used herein, the terms “cis-cleavage”, “cis-endonuclease activity”, “cis-mediated endonuclease activity”, “cis-nuclease activity”, “cis-mediated nuclease activity”, and variations thereof refer to sequence-specific cleavage of a target nucleic acid of interest, including an unblocked nucleic acid molecule or synthesized activating molecule, by a nucleic acid-guided nuclease in an RNP complex. Cis-cleavage is a single turn-over cleavage event in that only one substrate molecule is cleaved per event.

The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGATCGATCGA-5′ [SEQ ID NO: 1] is 100% complementary to a region of the nucleotide sequence 5′-GCTAGCTAGC-3′ [SEQ ID NO: 2].

As used herein, the term “contacting” refers to placement of two moieties in direct physical association, including in solid or liquid form. Contacting can occur in vitro with isolated cells (for example in a tissue culture dish or other vessel) or in vivo by administering an agent to a subject.

A “control” is a reference standard of a known value or range of values.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a crRNA region or guide sequence capable of hybridizing to the target strand of a target nucleic acid of interest, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease. The crRNA region of the gRNA is a customizable component that enables specificity in every nucleic acid-guided nuclease reaction. A gRNA can include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest to hybridize with the target nucleic acid of interest and to direct sequence-specific binding of a ribonucleoprotein (RNP) complex containing the gRNA and nucleic acid-guided nuclease to the target nucleic acid. Target nucleic acids of interest may include a protospacer adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region on the target nucleic acid of interest, including on an unblocked nucleic acid molecule or synthesized activating molecule. A gRNA may contain a spacer sequence including a plurality of bases complementary to a protospacer sequence in the target nucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its corresponding target nucleic acid of interest. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. A guide RNA may be from about 20 nucleotides to about 300 nucleotides long. Guide RNAs may be produced synthetically or generated from a DNA template.

“Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, a nucleic acid molecule (for example, a blocked nucleic acid molecule) may be modified by the introduction of non-natural nucleosides, nucleotides, and/or internucleoside linkages. In another embodiment, a modified protein (e.g., a nucleic acid-guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype.

The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. In some embodiments, the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5):1792-1797 (2004)). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, in embodiments, percent sequence identity values are generated using the sequence comparison computer program BLAST (Altschul et al., J. Mol. Biol., 215:403-410 (1990)).

As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complex containing a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest, guides the RNP to the target nucleic acid of interest and hybridizes to it. The hybridized target nucleic acid-gRNA units are cleaved by the nucleic acid-guided nuclease. In the cascade assays described herein, a first ribonucleoprotein complex (RNP1) includes a first guide RNA (gRNA) specific to a nucleic acid target nucleic acid of interest, and a first nucleic acid-guided nuclease, such as, for example, cas12a or cas14a for a DNA target nucleic acid, or cas13a for an RNA target nucleic acid. A second ribonucleoprotein complex (RNP2) for signal amplification includes a second guide RNA specific to an unblocked nucleic acid or synthesized activating molecule, and a second nucleic acid-guided nuclease, which may be different from or the same as the first nucleic acid-guided nuclease.

As used herein, the terms “protein” and “polypeptide” are used interchangeably. Proteins may or may not be made up entirely of amino acids.

As used herein, the term “sample” refers to tissues; cells or component parts; body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood; food; agricultural products; pharmaceuticals; cosmetics, nutriceuticals; personal care products; environmental substances such as soil, water, or air; industrial sites and products; and chemicals and compounds. A sample further may include a homogenate, lysate or extract. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

The terms “target DNA sequence”, “target sequence”, “target nucleic acid of interest”, “target molecule of interest”, “target nucleic acid”, or “target of interest” refer to any locus that is recognized by a gRNA sequence in vitro or in vivo. The “target strand” of a target nucleic acid of interest is the strand of the double-stranded target nucleic acid that is complementary to a gRNA. The spacer sequence of a gRNA may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or more complementary to the target nucleic acid of interest. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Full complementarity is not necessarily required provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of an RNP complex. A target nucleic acid of interest can include any polynucleotide, such as DNA (ssDNA or dsDNA) or RNA polynucleotides. A target nucleic acid of interest may be located in the nucleus or cytoplasm of a cell such as, for example, within an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast, or it can be exogenous to a host cell, such as a eukaryotic cell or a prokaryotic cell. The target nucleic acid of interest may be present in a sample, such as a biological or environmental sample, and it can be a viral nucleic acid molecule, a bacterial nucleic acid molecule, a fungal nucleic acid molecule, or a polynucleotide of another organism, such as a coding or a non-coding sequence, and it may include single-stranded or double-stranded DNA molecules, such as a cDNA or genomic DNA, or RNA molecules, such as mRNA, tRNA, and rRNA. The target nucleic acid may be associated with a protospacer adjacent motif (PAM) sequence, which may include a 2-5 base pair sequence adjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids can be detected by the disclosed method.

As used herein, the terms “trans-cleavage”, “trans-endonuclease activity”, “trans-mediated endonuclease activity”, “trans-nuclease activity”, “trans-mediated nuclease activity”, and variations thereof, refer to indiscriminate, non-sequence-specific cleavage of a nucleic acid molecule by an endonuclease (such as by a Cas12, Cas13, and Cas14) which is triggered by cis- (sequence-specific) cleavage. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved after initiation by a single turn-over cis-cleavage event.

Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR/Cas effector nucleases such as, but not limited to, engineered Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), CasY (Cas12d), Cas 13a nucleases or naturally-occurring proteins, such as a Cas12a isolated from, for example, Francisella tularensis subsp. novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (Gene ID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID: 15139718), and [Eubacterium] eligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein.

The term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many if not most regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Variants include modifications—including chemical modifications—to one or more amino acids that do not involve amino acid substitutions, additions or deletions.

A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter, methods, and cascade assays for detecting nucleic acids. The cascade assays described herein comprise first and second ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep the second ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex. The methods comprise the steps of providing cascade assay components, contacting the cascade assay components with a sample, and detecting a signal that is generated only when a target nucleic acid of interest is present in the sample ids.

Early and accurate detection and determination of infections and diseases is crucial for appropriate prevention strategies, accurate testing, confirmation, and further diagnosis and treatment. Nucleic acid-guided nucleases, such as the Cas12a endonuclease, can be utilized as diagnostic tools for the detection of target nucleic acids of interest associated with diseases. However, currently available state-of-the-art CRISPR Cas12a-based nucleic acid detection relies on DNA amplification before using Cas12a enzymes, which significantly hinders the ability to perform rapid point-of-care testing. The lack of rapidity is due to the fact that target-specific activation of Cas12a enzymes, referred herein as cis-cleavage, is a single turnover event in which the number of activated enzyme complexes is, at most, equal to the number of copies of the target nucleic acids of interest in the sample. Once a ribonucleoprotein (RNP) complex is activated after completion of cis-cleavage, the RNP complex initiates rapid non-specific trans-endonuclease activity, which is a multi-turnover event. Some currently available methods use trans-cleavage to cleave fluorescent reporters that are initially quenched to generate a signal, thereby indicating the presence of a cis-cleavage event—the target nucleic acid. However, the K_cat of activated Cas12a complex is 17/sec and 3/sec for dsDNA and ssDNA targets, respectively. Therefore, for less than 10,000 target copies, the number of reporters cleaved is not sufficient to generate a signal in less than 60 minutes. Hence, all current technologies rely on DNA amplification to first generate billions of target copies to activate a proportional number of ribonucleoprotein complexes to generate a detectable signal in 30-60 minutes.

The present disclosure describes a nucleic acid-guided nuclease cascade assay that can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. As described in detail below, the nucleic acid-guided nuclease cascade assays utilize signal amplification mechanisms comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules or blocked primer molecules, reporter moieties, and, in some embodiments, polymerases. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA (gRNA1) in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected. In this sense, the cascade assay is modular.

FIG. 1A provides a simplified diagram demonstrating a prior art method (1) of a nucleic acid-guided nuclease detection assay where target nucleic acids of interest from a sample must be amplified in order to be detected. First, assuming the presence of a target nucleic acid of interest in a sample, the target nucleic acid of interest (2) is amplified to produce many copies of the target nucleic acid of interest (4). The detection assay is initiated (step 2) when the target nucleic acid of interest (4) is combined with and binds to a pre-assembled ribonucleoprotein complex (6), which is part of a reaction mix. The ribonucleoprotein complex (6) comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides the RNP complex to the target nucleic acid of interest and hybridizes to it thereby activating the ribonucleoprotein complex (6). The nucleic acid-guided nuclease exhibits (i.e., possesses) both cis- and trans-cleavage activity, where trans-cleavage activity is initiated by cis-cleavage activity. Cis-cleavage activity occurs as the target nucleic acid of interest binds to the gRNA and is cleaved by the nucleic acid guided nuclease (i.e., activation). Once cis-cleavage of the target nucleic acid of interest is initiated, trans-cleavage activity is triggered, where trans-cleavage activity is indiscriminate, non-sequence-specific cleavage of nucleic acid molecules in the sample and is a multi-turnover event.

In step 3, the trans-cleavage activity triggers activation of reporter moieties (12) that are present in the reaction mix. The reporter moieties (12) may be a synthetic molecule linked or conjugated to a quencher (14) and a fluorophore (16) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (14) and fluorophore (16) typically are about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties (12) are described in greater detail below. As more activated ribonucleoprotein complexes (6) are activated (6→8), more trans-cleavage activity of the nucleic acid-guided nuclease in the ribonucleoprotein complex is activated and more reporter moieties are activated (where here, “activated” means unquenched); thus, the binding of the target nucleic acid of interest (4).

As noted above, the downside to the prior art, currently available state-of-the-art nucleic acid-guided nuclease detection assays is that these detection assays rely on DNA amplification, which, in addition to issues with multiplexing, significantly hinders the ability to perform rapid point-of-care testing. The lack of rapidity is due to cis-cleavage of a target nucleic acid of interest being a single turnover event in which the number of activated enzyme complexes is, at most, equal to the number of copies of the target nucleic acids of interest in the sample. Once the ribonucleoprotein complex is activated after completion of cis-cleavage, trans-cleavage activity of the reporter moieties that are initially quenched is generated. However, the K_cat of, e.g., activated Cas12a complex is 17/sec and 3/sec for dsDNA and ssDNA targets, respectively. Therefore, for less than 10,000 target copies, the number of reporters cleaved is not sufficient to generate a signal in less than 30-60 minutes.

FIG. 1B provides a simplified diagram demonstrating a method (100) of a nucleic acid-guided nuclease cascade assay. The cascade assay is initiated when the target nucleic acid of interest (104) binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides an RNP complex to the target nucleic acid of interest and hybridizes to it. Typically, preassembled RNP complexes are employed in the reaction mix—as opposed to separate nucleic acid-guided nucleases and gRNAs—to facilitate rapid detection of the target nucleic acid(s) of interest.

“Activation” of RNP1 refers to activating trans-cleavage activity of the nucleic acid-guided nuclease in RNP1 (106) by first initiating cis-cleavage where the target nucleic acid of interest is cut by the nucleic acid-guided nuclease. The cis-cleavage activity initiates trans-cleavage activity (i.e., multi-turnover activity) of the nucleic acid-guided nuclease, where trans-cleavage is indiscriminate, non-sequence-specific cutting of nucleic acid molecules by the nucleic acid-guided nuclease of RNP1 (102). This trans-cleavage activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (108) in various ways, which are described in detail below. Each newly activated RNP2 (110) activates more RNP2 (108→110), which in turn cleave reporter moieties (112). The reporter moieties (112) may be a synthetic molecule linked or conjugated to a quencher (114) and a fluorophore (116) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (114) and fluorophore (116) can be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties also are described in greater detail below. As more RNP2s are activated (108→110), more trans-cleavage activity is activated and more reporter moieties are activated (where here, “activated” means unquenched); thus, the binding of the target nucleic acid of interest (104) to RNP1 (102) initiates what becomes a cascade of signal production (120), which increases exponentially. The cascade assay thus comprises a single turnover event that triggers a multi-turnover event that then triggers another multi-turnover event. As described below in relation to FIG. 4, the reporter moieties (112) may be provided as molecules that are separate from the other components of the nucleic acid-guided nuclease cascade assay, or the reporter moieties may be covalently or non-covalently linked to the blocked nucleic acid molecules or synthesized activating molecules (i.e., the target molecules for the RNP2). The various components common to the embodiments of the cascade assay and methods described herein are described below.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule. Target nucleic acids of interest may be isolated from a sample or organism by standard laboratory techniques or may be synthesized by standard laboratory techniques (e.g., RT-PCR). In some embodiments, the target nucleic acids of interest are identified in a sample, such as a biological sample from a subject or an environmental sample (e.g., water or soil). Non-limiting examples of biological samples include blood, serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, a cell culture, and tissue. The source of the sample could be any mammal, such as, but not limited to, a human, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep, and bat. Samples may also be obtained from any other source, such as air, water, soil, surfaces, food, beverages, nutraceuticals, clinical sites or products, industrial sites and products, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples.

In some embodiments, the target nucleic acid of interest is from an infectious agent (e.g., a bacteria, protozoan, insect, worm, virus, or fungus). As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from bacteria, such as Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratia marcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum), Ureaplasma urealyticum, Mycoplasma genitalium, and/or Gardnerella vaginalis. As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a virus, such as adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus 0C43, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, herpes simplex virus 1, herpes simplex virus 2, human immunodeficiency virus (HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and/or human parvovirus B19 (B19V). Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a fungus, such as Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and/or Cryptococcus gattii. As another non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a protozoan, such as Trichomonas vaginalis. In some embodiments, other target nucleic acids of interest may be for non-infectious conditions, e.g., to be used for genotyping. Other target nucleic acids of interest and samples are described herein.

The cascade assays described herein are particularly well-suited for syndromic testing. Syndromic testing allows simultaneous testing for multiple causative agents that cause similar symptoms. Syndromic testing allows rapid triage of patients, such as those needing emergency care, those amenable to treatment with pharmaceutical agents, those needing to be quarantined, etc. In syndrome testing, multiple target nucleic acids of interest are pooled into a single reaction, and this process may be repeated in multiple, separate reactions. A positive result in one of the reactions indicates that one of the target nucleic acids of interest in that pool is present. Pools of two to 10,000 target nucleic acids of interest may be employed, e.g., 2-1000, 2-100, 2-50, or 2-10. Further testing may be used to identify the specific member of the pool, if warranted. Syndromic testing allows the rapid triage of patients with the ability to focus further care rapidly.

While the methods described herein do not require the target nucleic acid of interest to be DNA (and in fact it is specifically contemplated that the target nucleic acid of interest may be RNA), it is understood by those in the field that a reverse transcription step to convert target RNA to cDNA may be performed prior to or while contacting the biological sample with the composition.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in the reaction mix, either provided as a protein, a coding sequence for the protein, or in a ribonucleoprotein (RNP) complex. In some embodiments, the one or more nucleic acid-guided nucleases in the reaction mix may be, for example, a Cas endonuclease. Any nucleic acid-guided nuclease having both cis- and trans-endonuclease activity may be employed, and the same nucleic acid-guided nuclease may be used for both RNPs or different nucleic acid-guided nucleases may be used in RNP1 and RNP2. Note that trans-cleavage activity is not triggered unless and until cis-cleavage activity (i.e., sequence specific activity) is initiated. Nucleic acid-guided nucleases include Type V and Type VI nucleic acid-guided nucleases, as well as nucleic acid-guided nucleases that comprise a RuvC nuclease domain or a RuvC-like nuclease domain but lack an HNH nuclease domain. Nucleic acid-guided nucleases with these properties are reviewed in Makarova and Koonin, Methods Mol. Biol., 1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology, 37:67-78 (2020) and updated databases of nucleic acid-guided nucleases and nuclease systems that include newly-discovered systems include BioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org); Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder (crispercas.i2bc.p aris-saclay.fr).

The type of nucleic acid-guided nuclease utilized in the method of detection depends on the type of target nucleic acid of interest to be detected. For example, a DNA nucleic acid-guided nuclease (e.g., a Cas12a, Cas14a, or Cas3) should be utilized if the target nucleic acid of interest is a DNA molecule, and an RNA nucleic acid-guided nuclease (e.g., Cas13a or Cas12g) should be utilized if the target nucleic acid of interest is an RNA molecule. Exemplary nucleic acid-guided nucleases include, but are not limited to, Cas RNA-guided DNA endonucleases, such as Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; Cas RNA-guided RNA endonucleases, such as Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b (e.g., CccaCas13b, PsmCas13b), and Cas12g; and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease with cis-cleavage activity and collateral trans-cleavage activity. In some embodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Cas nuclease, such as a Cas12a, Cas13a, or Cas14a. In some embodiments, the nucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleic acid-guided nucleases may also be employed.

Guide RNA (gRNA)

The present disclosure detects a target nucleic acid of interest via a reaction mixture containing at least two gRNAs. Suitable guide RNAs include at least one crRNA region to enable specificity in every reaction. The gRNA of RNP1 is specific to a target nucleic acid of interest, and the gRNA of RNP2 is specific to an unblocked nucleic acid or a synthesized activating molecule (both described in detail herein). As will be clear given the description below, an advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (i.e., the gRNA specific to the target nucleic acid of interest), the cascade assay components can stay the same no matter what target nucleic acid(s) of interest are being detected. In this sense, the cascade assay is modular.

Like the nucleic acid-guided nuclease, the gRNA may be provided in the cascade assay reaction mix in a preassembled RNP, as an RNA molecule, or may also be provided as a DNA sequence to be transcribed, in, e.g., a vector backbone. If provided as a gRNA molecule, the gRNA sequence may include multiple endoribonuclease recognition sites (e.g., Csy4) for multiplex processing. Alternatively, if provided as a DNA sequence to be transcribed, an endoribonuclease recognition site is encoded between neighboring gRNA sequences and more than one gRNA can be transcribed in a single expression cassette. Direct repeats can also serve as endoribonuclease recognition sites for multiplex processing. Guide RNAs are generally about 20 nucleotides to about 300 nucleotides in length and may contain a spacer sequence containing a plurality of bases and complementary to a protospacer sequence in the target sequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its intended target nucleic acid of interest.

The gRNA of RNP1 is capable of complexing with the nucleic acid-guided nuclease to perform cis-cleavage of a target nucleic acid of interest (e.g., a DNA or RNA), which triggers non-sequence specific trans-cleavage of other molecules in the reaction mix. Guide RNAs include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest (or target sequences generated by unblocking blocked nucleic acid molecules or target sequences generated by synthesizing activating molecules as described below). Target sequences may include a protospacer-adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region of the target sequence.

In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistant circular molecule that can include several DNA bases between the 5′ end and the 3′ end of a natural guide RNA and is capable of binding a target sequence. The length of the circularized guide for RNP1 can be such that the circular form of guide can be complexed with a nucleic acid-guided nuclease to form a modified RNP1 which can still retain its cis- cleavage (specific) and trans-cleavage (non-specific) nuclease activity.

In any of the foregoing embodiments, the gRNA may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the gRNAs of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). By way of further example, a modified nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the assay “reaction mix” may comprise separate nucleic acid-guided nucleases and gRNAs (or coding sequences therefor), the cascade assays preferably comprise preassembled ribonucleoprotein complexes (RNPs) in the reaction mix, allowing for faster detection kinetics. The present cascade assay employs at least two types of RNP complexes, RNP1 and RNP2, each type containing a nucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the same nucleic acid-guided nuclease or may comprise different nucleic acid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are different and are configured to detect different nucleic acids. In some embodiments, the reaction mixture contains about 1 fM to about 10 μM of a given RNP1, or about 1 μM to about 1 μM of a given RNP1, or about 10 μM to about 500 μM of a given RNP1. In some embodiments the reaction mixture contains about 6×10⁴ to about 6×10¹² complexes per microliter (μl) of a given RNP1, or about 6×10⁶ to about 6×10¹⁰ complexes per microliter (μl) of a given RNP1. In some embodiments, the reaction mixture contains about 1 fM to about 1 mM of a given RNP2, or about 1 μM to about 500 μM of a given RNP2, or about 10 μM to about 100 μM of a given RNP2. In some embodiments the reaction mixture contains about 6×10⁴ to about 6×10¹⁴ complexes per microliter (μl) of a given RNP2 or about 6×10⁶ to about 6×10¹² complexes per microliter (μl) of a given RNP2. (See Example II below describing preassembling RNPs and Examples V-IX below describing various cascade assay conditions, including performing the cascade assay at room temperature.)

In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,0000 RNP1s), where different RNP1s comprise a different gRNA (or crRNA thereof) polynucleotide sequence. For example, a reaction mixture designed for syndromic testing by definition comprises more than one unique RNP1-gRNA (or RNP1-crRNA) ribonucleoprotein complex for the purpose of detecting more than one target nucleic acid of interest. More than one RNP1 may also be present for the purpose of targeting more than one target nucleic acid of interest from a single organism or condition.

In any of the foregoing embodiments, the gRNA of RNP1 may be homologous or heterologous, relative to the gRNA of other RNP1 present in the reaction mixture. A homologous mixture of RNP1 gRNAs has a number of gRNAs with the same nucleotide sequence, whereas a heterologous mixture of RNP1 gRNAs has multiple gRNAs with different nucleotide sequences (e.g., gRNAs targeting different loci, genes, variants, and/or microbial species). Therefore, the disclosed methods of identifying one or more target nucleic acids of interest may include a reaction mixture containing more than two heterologous gRNAs, more than three heterologous gRNAs, more than four heterologous gRNAs, more than five heterologous gRNAs, more than six heterologous gRNAs, more than seven heterologous gRNAs, more than eight heterologous gRNAs, more than nine heterologous gRNAs, more than ten heterologous gRNAs, more than eleven heterologous gRNAs, more than twelve heterologous gRNAs, more than thirteen heterologous gRNAs, more than fourteen heterologous gRNAs, more than fifteen heterologous gRNAs, more than sixteen heterologous gRNAs, more than seventeen heterologous gRNAs, more than eighteen heterologous gRNAs, more than nineteen heterologous gRNAs, more than twenty heterologous gRNAs, more than twenty-one heterologous gRNAs, more than twenty-three heterologous gRNAs, more than twenty-four heterologous gRNAs, or more than twenty-five heterologous gRNAs. Such a heterologous mixture of RNP1 gRNAs in a single reaction enables the capability of syndromic testing.

As a first non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP 1 s having a gRNA targeting parainfluenza virus 1; a number of RNP1s having a gRNA targeting human metapneumovirus; a number of RNP1s having a gRNA targeting human rhinovirus; a number of RNP 1 s having a gRNA targeting human enterovirus; and a number of RNP 1 s having a gRNA targeting coronavirus HKU1. As a second non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP1s containing a gRNA targeting two or more SARS-Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariants thereof.

Reporter Moieties

The cascade assay detects a target nucleic acid of interest via detection of a signal generated in the reaction mix by a reporter moiety. In some embodiments the detection of the target nucleic acid of interest occurs in about 10 minutes or less (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute or less; e.g., FIGS. 6-9, and in some embodiments the detection of the target nucleic acid molecule is in about 5 minutes or less (e.g., 5, 4, 3, 2, or 1 minute or less; e.g., FIGS. 10-14). In some embodiments, the detection of the target nucleic acid molecule is in about 1 minute (e.g., FIGS. 10-13).

Depending on the type of reporter moiety used, trans- and/or cis-cleavage by the nucleic acid-guided nuclease in RNP2 releases a signal. In some embodiments, trans-cleavage of stand-alone (e.g., not bound to any blocked nucleic acid molecules) reporter moieties may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time (shown in FIG. 1B and at top of FIG. 4). Trans-cleavage by either an activated RNP1 or an activated RNP2 may release a signal. In alternative embodiments, the reporter moiety may be bound to the blocked nucleic acid molecule, where trans-cleavage of the blocked nucleic acid molecule and conversion to an unblocked nucleic acid molecule may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time, thus allowing for real time reporting of results (shown at FIG. 4, center). In yet another embodiment, the reporter moiety may be bound to a blocked nucleic acid molecule such that cis-cleavage following the binding of the RNP2 to an unblocked nucleic acid molecule releases a PAM distal sequence, which in turn generates a signal at rates that are proportional to the cleavage rate (shown at FIG. 4, bottom). In this case, activation of RNP2 by cis- (target specific) cleavage of the unblocked nucleic acid molecule directly produces a signal, rather than producing a signal via indiscriminate trans-cleavage activity. Alternatively. or in addition, the reporter moiety may be bound to the gRNA.

The reporter moiety may be a synthetic molecule linked or conjugated to a reporter and quencher such as, for example, a TaqMan probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The reporter and quencher may be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Alternatively, signal generation may occur through different mechanisms. Other detectable moieties, labels, or reporters can also be used to detect a target nucleic acid of interest as described herein. Reporter moieties can be labeled in a variety of ways, including direct or indirect attachment of a detectable moiety such as a fluorescent moiety, hapten, or colorimetric moiety. Examples of detectable moieties include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, and protein-protein binding pairs, e.g., protein-antibody binding pairs. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, and phycoerythrin. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorinidases, phosphatases, peroxidases, and cholinesterases. Identifiable markers also include radioactive elements such as ¹²⁵1, ³⁵S, ¹⁴C, or ³H.

The methods used to detect the generated signal will depend on the reporter moiety or moieties used. For example, a radioactive label can be detected using a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Fluorescent labels can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Simple colorimetric labels can be detected by observing the color associated with the label. When pairs of fluorophores are used in an assay, fluorophores are chosen that have distinct emission patterns (wavelengths) so that they can be easily distinguished. In some embodiments, the signal can be detected by lateral flow assays (LFAs). Lateral flow tests are simple devices intended to detect the presence or absence of a target nucleic acid of interest in a sample. LFAs can use nucleic acid molecules conjugated nanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detection probe, which hybridizes to a complementary target sequence. (See FIGS. 5A and 5B and the description thereof below.) The classic example of an LFA is the home pregnancy test.

Single-stranded nucleic acid reporter moieties such as ssDNA reporter moieties or RNA molecules can be introduced to show a signal change proportional to the cleavage rate, which increases with every new activated RNP2 complex over time. In some embodiments and as described in detail below, single-stranded nucleic acid reporter moieties can also be embedded into the blocked nucleic acid molecules for real time reporting of results.

For example, the method of detecting a target nucleic acid molecule in a sample using a cascade assay as described herein can involve contacting the reaction mix with a labeled detection ssDNA containing a fluorescent resonance energy transfer (FRET) pair, a quencher/phosphor pair, or both. A FRET pair consists of a donor chromophore and an acceptor chromophore, where the acceptor chromophore may be a quencher molecule. FRET pairs (donor/acceptor) suitable for use include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL, fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, and fluorescein/LC Red 705. In addition, a fluorophore/quantum dot donor/acceptor pair can be used. EDANS is (5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is 5({2-[(iodoacetyl)amino] ethyl} amino)naphthalene-1-sulfonic acid); DABCYL is 4-(4-dimethylaminophenyl) diazenylbenzoic acid. Useful quenchers include, but are not limited to, DABCYL, QSY 7 and QSY 33.

In any of the foregoing embodiments, the reporter moiety may comprise one or more modified nucleic acid molecules, containing a modified nucleoside or nucleotide. In some embodiments the modified nucleoside or nucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, template molecules, synthesized activating molecules, and reporter moieties), the nucleic acid molecules may be used in a wholly or partially modified form. Typically, modifications to the blocked nucleic acids, gRNAs, template molecules, reporter moieties, and blocked primer molecules described herein are introduced to optimize the molecule's biophysical properties (e.g., increasing endonuclease resistance and/or increasing thermal stability). Modifications typically are achieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages.

For example, one or more of the cascade assay components may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, reporter molecules, synthesized activating molecules, and template molecules) may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and/or 2-pyridone. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in U.S. Pat. No. 3,687,808; Kroschwitz, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); and Sanghvi, Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed., 1993, pp. 289-302.

In addition to or as an alternative to nucleoside modifications, the cascade assay components may comprise 2′ sugar modifications, including 2′-O-methyl (2′-O-Me), 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and/or 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify the nucleic acid molecules described herein (i.e., blocked nucleic acids, gRNAs, synthesized activating molecules, reporter molecules, and blocked primer molecules) may include all possible orientations of OH; F; O—, S—, or N-alkyl (mono- or di-); O—, S—, or N-alkenyl (mono- or di-); O—, S— or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Finally, modifications to the cascade assay components may comprise internucleoside modifications such as phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Cascade Assay Employing Blocked Nucleic Acids

FIG. 1B depicts the cascade assay generally. A specific embodiment of the cascade assay utilizing blocked nucleic acids is depicted in FIG. 2A. In this embodiment, a blocked nucleic acid is used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method in FIG. 2A begins with providing the cascade assay components RNP1 (201), RNP2 (202) and blocked nucleic acid molecules (203). RNP1 (201) comprises a gRNA specific for a target nucleic acid of interest and a nucleic acid-guided nuclease (e.g., Cas 12a or Cas 14 for a DNA target nucleic acid of interest or a Cas 13a for an RNA target nucleic acid of interest) and RNP2 (202) comprises a gRNA specific for an unblocked nucleic acid molecule and a nucleic acid-guided nuclease (again, Cas 12a or Cas 14 for a DNA unblocked nucleic acid molecule or a Cas 13a for an RNA unblocked nucleic acid molecule). As described above, the nucleic acid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same or different depending on the type of target nucleic acid of interest and unblocked nucleic acid molecule. What is key, however, is that the nucleic acid-guided nucleases in RNP1 and RNP2 may be activated to have trans-cleavage activity following initiation of cis-cleavage activity.

In a first step, a sample comprising a target nucleic acid of interest (204) is added to the cascade assay reaction mix. The target nucleic acid of interest (204) combines with and activates RNP1 (205) but does not interact with or activate RNP2 (202). Once activated, RNP1 cuts the target nucleic acid of interest (204) via sequence-specific cis-cleavage, which then activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including the blocked nucleic acid molecules (203). At least one of the blocked nucleic acid molecules (203) becomes an unblocked nucleic acid molecule (206) when the blocking moiety (207) is removed. As described below, “blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and/or structural modifications.

Once at least one of the blocked nucleic acid molecules (203) is unblocked, the unblocked nucleic acid molecule (206) can then interact with and activate an RNP2 (208) complex. Because the nucleic acid-guided nucleases in the RNP1×(205) and RNP2×(208) have both cis- and trans-cleavage activity, more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering activation of more RNP2 (208) complexes and more trans-cleavage activity in a cascade. FIG. 2A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (209) comprise a quencher (210) and a fluorophore (211) linked by a nucleic acid sequence. As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (205) and RNP2 (208). The intact reporter moieties (209) become activated reporter moieties (212) when the quencher (210) is separated from the fluorophore (211), emitting a fluorescent signal (213). Signal strength increases rapidly as more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering cis-cleavage activation of more RNP2s (208) and thus more trans-cleavage activity of the reporter moieties (209). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4. One particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (gRNA1), the cascade assay components are modular in the sense that the components stay the same no matter what target nucleic acid(s) of interest are being detected.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule (220) and an exemplary technique for unblocking the blocked nucleic acid molecules described herein. A blocked single-stranded or double-stranded, circular or linear, DNA or RNA molecule (220) comprising a target strand (222) may contain a partial hybridization with a complementary non-target strand nucleic acid molecule (224) containing unhybridized and cleavable secondary loop structures (226) (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Trans-cleavage of the loops by, e.g., activated RNP1s or RNP2s, generates short strand nucleotide sequences (228) which, because of the short length and low melting temperature T_m, can dehybridize at room temperature (e.g., 15°-25° C.), thereby unblocking the blocked nucleic acid molecule (220) to create an unblocked nucleic acid molecule (230), enabling the internalization of the unblocked nucleic acid molecule (230) (target strand) into an RNP2, leading to RNP2 activation.

A blocked nucleic acid molecule may be single-stranded or double-stranded, circular or linear, and may further contain a partially hybridized nucleic acid sequence containing cleavable secondary loop structures, as exemplified by “L” in FIGS. 2C-2E. Such blocked nucleic acids typically have a low binding affinity, or high dissociation constant (K_d) in relation to binding to RNP2 and may be referred to herein as a high K_d nucleic acid molecule. In the context of the present disclosure, the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low K_d values range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_d values are in the range of 100 nM to about 100 μM (10 mM) and thus are about 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹- to 10¹⁰-fold or higher as compared to low K_d values.

The blocked nucleic acid molecules (high K_d molecules) described herein can be converted into unblocked nucleic acid molecules (low K_d molecules—also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNP1s and RNP2s). The unblocked nucleic acid molecule has a higher binding affinity for the gRNA in the RNP2 than does the blocked nucleic acid molecule, although there may be some “leakiness” where some blocked nucleic acid molecules are able to interact with the gRNA in the RNP2. However, an unblocked nucleic acid molecule has a substantially higher likelihood than a blocked nucleic acid molecule to hybridize with the gRNA of RNP2.

Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2 activation triggers trans-cleavage activity, which in turn leads to more RNP2 activation by further cleaving blocked nucleic acid molecules, resulting in a positive feedback loop.

In embodiments where blocked nucleic acid molecules are linear and/or form a secondary structure, the blocked nucleic acid molecules may be single-stranded (ss) or double-stranded (ds) and contain a first nucleotide sequence and a second nucleotide sequence. The first nucleotide sequence has sufficient complementarity to hybridize to a gRNA of RNP2, and the second nucleotide sequence does not. The first and second nucleotide sequences of a blocked nucleic acid molecule may be on the same nucleic acid molecule (e.g., for single-strand embodiments) or on separate nucleic acid molecules (e.g., for double strand embodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the second nucleotide sequence converts the blocked nucleic acid molecule to a single-strand unblocked nucleic acid molecule. The unblocked nucleic acid molecule contains only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-endonuclease activity of RNP2.

In some embodiments, the second nucleotide sequence at least partially hybridizes to the first nucleotide sequence, resulting in a secondary structure containing at least one loop (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Such loops block the nucleic acid molecule from binding or incorporating into an RNP complex in a manner to initiate trans cleavage (see, e.g., the exemplary structures in FIGS. 2C-2E).

In some embodiments, the blocked nucleic acid molecule may contain a protospacer adjacent motif (PAM) sequence, or partial PAM sequence, positioned between the first and second nucleotide sequences, where the first sequence is 5′ to the PAM sequence, or partial PAM sequence, (see FIG. 2G). Inclusion of a PAM sequence may increase the reaction kinetics internalizing the unblocked nucleic acid molecule into RNP2 and thus decrease the time to detection. In other embodiments, the blocked nucleic acid molecule does not contain a PAM sequence.

In some embodiments, the blocked nucleic acid molecules (i.e., high K_d nucleic acid molecules—in relation to binding to RNP2) of the disclosure may include a structure represented by Formula I (e.g., FIG. 2C), Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or Formula IV (e.g., FIG. 2F) wherein Formulas I-IV are in the 5′-to-3′ direction:

A-(B-L)J-C-M-T-D  (Formula I);

• • wherein A is 0-15 nucleotides in length;
  • B is 4-12 nucleotides in length;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10;
  • C is 4-15 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands;
  • T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25) and comprises a sequence complementary to B and C; and
  • D is 0-10 nucleotides in length and comprises a sequence complementary to A;

D-T-T′-C-(L-B)J-A  (Formula II);

• • wherein D is 0-10 nucleotides in length;
  • T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
  • T′ is 1-10 nucleotides in length and does not hybridize with T;
  • C is 4-15 nucleotides in length and comprises a sequence complementary to T;
  • L is 3-25 nucleotides in length and does not hybridize with T;
  • B is 4-12 nucleotides in length and comprises a sequence complementary to T;
  • J is an integer between 1 and 10;
  • A is 0-15 nucleotides in length and comprises a sequence complementary to D;

T-D-M-A-(B-L)J-C  (Formula III);

wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25);

• • D is 0-10 nucleotides in length;
  • M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and
  • A-(B-L)J-C are separate nucleic acid strands;
  • A is 0-15 nucleotides in length and comprises a sequence complementary to D;
  • B is 4-12 nucleotides in length and comprises a sequence complementary to T;
  • L is 3-25 nucleotides in length;
  • J is an integer between 1 and 10; and
  • C is 4-15 nucleotides in length;

T-D-M-A-Lp-C  (Formula IV);

• • wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
  • D is 0-15 nucleotides in length;
  • M is 1-25 nucleotides in length;
  • A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
  • L is 3-25 nucleotides in length;
  • p is 0 or 1;
  • C is 4-15 nucleotides in length and comprises a sequence complementary to T.
    In alternative embodiments of any of these molecules, T (or T-T′) can have a maximum length of 1000 nucleotides, e.g., at most 200, at most 135, at most 75, at most 50, or at most 25.

Nucleotide mismatches can be introduced in any of the above structures containing double strand segments (for example, where M is absent in Formula I or Formula III) to reduce the melting temperature (Tm) of the segment such that once the loop (L) is cleaved, the double strand segment is unstable and dehybridizes rapidly. The percentage of nucleotide mismatches of a given segment may vary between 0% and 50%; however, the maximum number of nucleotide mismatches is limited to a number where the secondary loop structure still forms. “Segments” in the above statement refers to A, B, and C. In other words, the number of hybridized bases can be less than or equal to the length of each double strand segment and vary based on number of mismatches introduced.

In any blocked nucleic acid molecule having the structure of Formula I, III, or IV, T will have sequence complementarity to a nucleotide sequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotide sequence of T is to be designed such that hybridization of T to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. In any blocked nucleic acid molecule having structure of Formula II, T-T′ will have sequence complementarity to a sequence (e.g., a spacer sequence) within the gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed such that hybridization of T-T′ to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. For T or T-T′, full complementarity to the gRNA is not necessarily required, provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of RNP2.

Exemplary nucleotide sequences of blocked nucleic acid molecules (e.g., SEQ ID NOs: 14-1421) include those in Table 1.

TABLE 1
Nucleotide sequences of blocked nucleic acid molecules.
SEQ ID NO:      Sequence
SEQ ID NO: 14   GATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA
                TATATATATATAGTATC
SEQ ID NO: 15   GACACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA
                TATATATATATAGTGTC
SEQ ID NO: 16   GATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA
                TATATATATATCGTATC
SEQ ID NO: 17   GGATCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA
                TATATATATATAGATCC
SEQ ID NO: 18   GACACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA
                TATATATATATCGTGTC
SEQ ID NO: 19   GGATCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA
                TATATATATATCGATCC
SEQ ID NO: 20   GCGTCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA
                TATATATATATAGACGC
SEQ ID NO: 21   GCGTCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA
                TATATATATATCGACGC
SEQ ID NO: 22   GTATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT
                ATATATATATAGTATAC
SEQ ID NO: 23   GTGATCTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT
                ATATATATATAGATCAC
SEQ ID NO: 24   GTATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT
                ATATATATATCGTATAC
SEQ ID NO: 25   GTATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT
                ATACATATATCGTATAC
SEQ ID NO: 26   GGATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT
                ATATATATATAGTATCC
SEQ ID NO: 27   GTGATCTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT
                ATATATATATCGATCAC
SEQ ID NO: 28   GTGATCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT
                ATACATATATCGATCAC
SEQ ID NO: 29   GGATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT
                ATATATATATCGTATCC
SEQ ID NO: 30   GGATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT
                ATACATATATCGTATCC
SEQ ID NO: 31   GCGATCTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT
                ATATATATATAGATCGC
SEQ ID NO: 32   GCGATCTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT
                ATATATATATCGATCGC
SEQ ID NO: 33   GCGATCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT
                ATACATATATCGATCGC
SEQ ID NO: 34   GATATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA
                TATATATATAGTATATC
SEQ ID NO: 35   GATATATTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                TATATATATCATATATC
SEQ ID NO: 36   GATATATTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                TACATATATCATATATC
SEQ ID NO: 37   GTGATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA
                TATATATATAGTATCAC
SEQ ID NO: 38   GATATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                TATATATATCGTATATC
SEQ ID NO: 39   GATATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                TACATATATCGTATATC
SEQ ID NO: 40   GGTATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA
                TATATATATAGTATACC
SEQ ID NO: 41   GTGATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                TATATATATCGTATCAC
SEQ ID NO: 42   GTGATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                TACATATATCGTATCAC
SEQ ID NO: 43   GGTATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                TATATATATCGTATACC
SEQ ID NO: 44   GGTATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                TACATATATCGTATACC
SEQ ID NO: 45   GGTGTACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA
                TATATATATAGTACACC
SEQ ID NO: 46   GGTGTACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                TATATATATCGTACACC
SEQ ID NO: 47   GGTGTACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                TACATATATCGTACACC
SEQ ID NO: 48   GTATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTAT
                ATATATATAGTATATAC
SEQ ID NO: 49   GTATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTAT
                ATATATATCGTATATAC
SEQ ID NO: 50   GTATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT
                ACATATATCGTATATAC
SEQ ID NO: 51   GTATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA
                TACATGATCGTATATAC
SEQ ID NO: 52   GTATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA
                TATATGATCGTATATAC
SEQ ID NO: 53   GGATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTAT
                ATATATATAGTATATCC
SEQ ID NO: 54   GGATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTAT
                ATATATATCGTATATCC
SEQ ID NO: 55   GGATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT
                ACATATATCGTATATCC
SEQ ID NO: 56   GGATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA
                TACATGATCGTATATCC
SEQ ID NO: 57   GGATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA
                TATATGATCGTATATCC
SEQ ID NO: 58   GGTGATACTTTTTATTTTTTATATATATATATTTTTATTTTTTAT
                ATATATATAGTATCACC
SEQ ID NO: 59   GGTGATACTTTTTATTTTTGATATATATATATTTTTATTTTTTAT
                ATATATATCGTATCACC
SEQ ID NO: 60   GGTGATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT
                ACATATATCGTATCACC
SEQ ID NO: 61   GGTGATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA
                TACATGATCGTATCACC
SEQ ID NO: 62   GGTGATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA
                TATATGATCGTATCACC
SEQ ID NO: 63   GGTGATCCTTTTTATTTTTTATATATATATATTTTTATTTTTTAT
                ATATATATAGGATCACC
SEQ ID NO: 64   GGTGATCCTTTTTATTTTTGATATATATATATTTTTATTTTTTAT
                ATATATATCGGATCACC
SEQ ID NO: 65   GGTGATCCTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT
                ACATATATCGGATCACC
SEQ ID NO: 66   GGTGATCCTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA
                TACATGATCGGATCACC
SEQ ID NO: 67   GGTGATCCTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA
                TATATGATCGGATCACC
SEQ ID NO: 68   GATATATCACTTTTTATTTTTTATATATATATTTTTATTTTTTAT
                ATATATAGTGATATATC
SEQ ID NO: 69   GTATATACATTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                ATATATCATGTATATAC
SEQ ID NO: 70   GTATATACATTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                ATATATCATGTATATAC
SEQ ID NO: 71   GTATATACATTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                CATGATCATGTATATAC
SEQ ID NO: 72   GTATATACATTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                TATGATCATGTATATAC
SEQ ID NO: 73   GGATATACACTTTTTATTTTTTATATATATATTTTTATTTTTTAT
                ATATATAGTGTATATCC
SEQ ID NO: 74   GGATATACATTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                ATATATCATGTATATCC
SEQ ID NO: 75   GGATATACATTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                ATATATCATGTATATCC
SEQ ID NO: 76   GGATATACATTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                CATGATCATGTATATCC
SEQ ID NO: 77   GGATATACATTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                TATGATCATGTATATCC
SEQ ID NO: 78   GGGTATATACTTTTTATTTTTTATATATATATTTTTATTTTTTAT
                ATATATAGTATATACCC
SEQ ID NO: 79   GGATATACACTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                ATATATCGTGTATATCC
SEQ ID NO: 80   GGATATACACTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                ATATATCGTGTATATCC
SEQ ID NO: 81   GGATATACACTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                CATGATCGTGTATATCC
SEQ ID NO: 82   GGATATACACTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                TATGATCGTGTATATCC
SEQ ID NO: 83   GGGTATATACTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                ATATATCGTATATACCC
SEQ ID NO: 84   GGGTATATACTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                ATATATCGTATATACCC
SEQ ID NO: 85   GGGTATATACTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                CATGATCGTATATACCC
SEQ ID NO: 86   GGGTATATACTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                TATGATCGTATATACCC
SEQ ID NO: 87   GGATGTACACTTTTTATTTTTTATATATATATTTTTATTTTTTAT
                ATATATAGTGTACATCC
SEQ ID NO: 88   GGATGTACACTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                ATATATCGTGTACATCC
SEQ ID NO: 89   GGATGTACACTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                ATATATCGTGTACATCC
SEQ ID NO: 90   GGATGTACACTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                CATGATCGTGTACATCC
SEQ ID NO: 91   GGATGTACACTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                TATGATCGTGTACATCC
SEQ ID NO: 92   GTATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT
                ATATATATATATATAGTATATAC
SEQ ID NO: 93   GTATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTT
                ATATATATATATATCGTATATAC
SEQ ID NO: 94   GGATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT
                ATATATATATATATAGTATATCC
SEQ ID NO: 95   GGATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTT
                ATATATATATATATCGTATATCC
SEQ ID NO: 96   GGTGATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT
                ATATATATATATATAGTATCACC
SEQ ID NO: 97   GGTGATACTTTTTATTTTTGATATATATATATATTTTTTATTTTT
                ATATATATATATATCGTATCACC
SEQ ID NO: 98   GGTGATCCTTTTTATTTTTTATATATATATATATTTTTTATTTTT
                ATATATATATATATAGGATCACC
SEQ ID NO: 99   GGTGATCCTTTTTATTTTTGATATATATATATATTTTTTATTTTT
                ATATATATATATATCGGATCACC
SEQ ID NO: 100  GATATATCACTTTTTATTTTTTATATATATATATATTTTTTATTT
                TTATATATATATATATAGTGATATATC
SEQ ID NO: 101  GTATATACATTTTTTATTTTTGATATATATATATATTTTTTATTT
                TTATATATATATATATCATGTATATAC
SEQ ID NO: 102  GGATATACACTTTTTATTTTTTATATATATATATATTTTTTATTT
                TTATATATATATATATAGTGTATATCC
SEQ ID NO: 103  GGATATACATTTTTTATTTTTGATATATATATATATTTTTTATTT
                TTATATATATATATATCATGTATATCC
SEQ ID NO: 104  GGGTATATACTTTTTATTTTTTATATATATATATATTTTTTATTT
                TTATATATATATATATAGTATATACCC
SEQ ID NO: 105  GGATATACACTTTTTATTTTTGATATATATATATATTTTTTATTT
                TTATATATATATATATCGTGTATATCC
SEQ ID NO: 106  GGGTATATACTTTTTATTTTTGATATATATATATATTTTTTATTT
                TTATATATATATATATCGTATATACCC
SEQ ID NO: 107  GTATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTT
                ATATATATATATAGTATATAC
SEQ ID NO: 108  GTATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTT
                ATATATATATATCGTATATAC
SEQ ID NO: 109  GTATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTT
                ATATACATATATCGTATATAC
SEQ ID NO: 110  GGATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTT
                ATATATATATATAGTATATCC
SEQ ID NO: 111  GGATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTT
                ATATATATATATCGTATATCC
SEQ ID NO: 112  GGATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTT
                ATATACATATATCGTATATCC
SEQ ID NO: 113  GGTGATACTTTTTATTTTTTATATATATATATATTTTTATTTTTT
                ATATATATATATAGTATCACC
SEQ ID NO: 114  GGTGATACTTTTTATTTTTGATATATATATATATTTTTATTTTTT
                ATATATATATATCGTATCACC
SEQ ID NO: 115  GGTGATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTT
                ATATACATATATCGTATCACC
SEQ ID NO: 116  GGTGATCCTTTTTATTTTTTATATATATATATATTTTTATTTTTT
                ATATATATATATAGGATCACC
SEQ ID NO: 117  GGTGATCCTTTTTATTTTTGATATATATATATATTTTTATTTTTT
                ATATATATATATCGGATCACC
SEQ ID NO: 118  GGTGATCCTTTTTATTTTTGATATATGTATATATTTTTATTTTTT
                ATATACATATATCGGATCACC
SEQ ID NO: 119  GATATATCACTTTTTATTTTTTATATATATATATATTTTTATTTT
                TTATATATATATATAGTGATATATC
SEQ ID NO: 120  GTATATACATTTTTTATTTTTGATATATATATATATTTTTATTTT
                TTATATATATATATCATGTATATAC
SEQ ID NO: 121  GTATATACATTTTTTATTTTTGATATATGTATATATTTTTATTTT
                TTATATACATATATCATGTATATAC
SEQ ID NO: 122  GGATATACACTTTTTATTTTTTATATATATATATATTTTTATTTT
                TTATATATATATATAGTGTATATCC
SEQ ID NO: 123  GGATATACATTTTTTATTTTTGATATATATATATATTTTTATTTT
                TTATATATATATATCATGTATATCC
SEQ ID NO: 124  GGATATACATTTTTTATTTTTGATATATGTATATATTTTTATTTT
                TTATATACATATATCATGTATATCC
SEQ ID NO: 125  GGGTATATACTTTTTATTTTTTATATATATATATATTTTTATTTT
                TTATATATATATATAGTATATACCC
SEQ ID NO: 126  GGATATACACTTTTTATTTTTGATATATATATATATTTTTATTTT
                TTATATATATATATCGTGTATATCC
SEQ ID NO: 127  GGATATACACTTTTTATTTTTGATATATGTATATATTTTTATTTT
                TTATATACATATATCGTGTATATCC
SEQ ID NO: 128  GGGTATATACTTTTTATTTTTGATATATATATATATTTTTATTTT
                TTATATATATATATCGTATATACCC
SEQ ID NO: 129  GGGTATATACTTTTTATTTTTGATATATGTATATATTTTTATTTT
                TTATATACATATATCGTATATACCC
SEQ ID NO: 130  GATATATCACTTTTTATTTTTTATATATATATATTTTTTATTTTT
                ATATATATATATAGTGATATATC
SEQ ID NO: 131  GTATATACATTTTTTATTTTTGATATATATATATTTTTTATTTTT
                ATATATATATATCATGTATATAC
SEQ ID NO: 132  GTATATACATTTTTTATTTTTGATATATGTATATTTTTTATTTTT
                ATATACATATATCATGTATATAC
SEQ ID NO: 133  GGATATACACTTTTTATTTTTTATATATATATATTTTTTATTTTT
                ATATATATATATAGTGTATATCC
SEQ ID NO: 134  GGATATACATTTTTTATTTTTGATATATATATATTTTTTATTTTT
                ATATATATATATCATGTATATCC
SEQ ID NO: 135  GGATATACATTTTTTATTTTTGATATATGTATATTTTTTATTTTT
                ATATACATATATCATGTATATCC
SEQ ID NO: 136  GGGTATATACTTTTTATTTTTTATATATATATATTTTTTATTTTT
                ATATATATATATAGTATATACCC
SEQ ID NO: 137  GGATATACACTTTTTATTTTTGATATATATATATTTTTTATTTTT
                ATATATATATATCGTGTATATCC
SEQ ID NO: 138  GGATATACACTTTTTATTTTTGATATATGTATATTTTTTATTTTT
                ATATACATATATCGTGTATATCC
SEQ ID NO: 139  GGGTATATACTTTTTATTTTTGATATATATATATTTTTTATTTTT
                ATATATATATATCGTATATACCC
SEQ ID NO: 140  GGGTATATACTTTTTATTTTTGATATATGTATATTTTTTATTTTT
                ATATACATATATCGTATATACCC
SEQ ID NO: 141  GATATATCACTTTTTATTTTTTATATATATATATTTTTATTTTTT
                ATATATATATAGTGATATATC
SEQ ID NO: 142  GTATATACATTTTTTATTTTTGATATATATATATTTTTATTTTTT
                ATATATATATCATGTATATAC
SEQ ID NO: 143  GTATATACATTTTTTATTTTTGATATATGTATATTTTTATTTTTT
                ATACATATATCATGTATATAC
SEQ ID NO: 144  GTATATACATTTTTTATTTTTGATCATGTATATTTTTTATTTTTA
                TATACATGATCATGTATATAC
SEQ ID NO: 145  GTATATACATTTTTTATTTTTGATCATATATGTTTTTTATTTTTA
                CATATATGATCATGTATATAC
SEQ ID NO: 146  GGATATACACTTTTTATTTTTTATATATATATATTTTTATTTTTT
                ATATATATATAGTGTATATCC
SEQ ID NO: 147  GGATATACATTTTTTATTTTTGATATATATATATTTTTATTTTTT
                ATATATATATCATGTATATCC
SEQ ID NO: 148  GGATATACATTTTTTATTTTTGATATATGTATATTTTTATTTTTT
                ATACATATATCATGTATATCC
SEQ ID NO: 149  GGATATACATTTTTTATTTTTGATCATGTATATTTTTTATTTTTA
                TATACATGATCATGTATATCC
SEQ ID NO: 150  GGATATACATTTTTTATTTTTGATCATATATGTTTTTTATTTTTA
                CATATATGATCATGTATATCC
SEQ ID NO: 151  GGGTATATACTTTTTATTTTTTATATATATATATTTTTATTTTTT
                ATATATATATAGTATATACCC
SEQ ID NO: 152  GGATATACACTTTTTATTTTTGATATATATATATTTTTATTTTTT
                ATATATATATCGTGTATATCC
SEQ ID NO: 153  GGATATACACTTTTTATTTTTGATATATGTATATTTTTATTTTTT
                ATACATATATCGTGTATATCC
SEQ ID NO: 154  GGATATACACTTTTTATTTTTGATCATGTATATTTTTTATTTTTA
                TATACATGATCGTGTATATCC
SEQ ID NO: 155  GGATATACACTTTTTATTTTTGATCATATATGTTTTTTATTTTTA
                CATATATGATCGTGTATATCC
SEQ ID NO: 156  GGGTATATACTTTTTATTTTTGATATATATATATTTTTATTTTTT
                ATATATATATCGTATATACCC
SEQ ID NO: 157  GGGTATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTT
                ATACATATATCGTATATACCC
SEQ ID NO: 158  GGGTATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTA
                TATACATGATCGTATATACCC
SEQ ID NO: 159  GGGTATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTA
                CATATATGATCGTATATACCC
SEQ ID NO: 160  GTACATATATTTTTTTATTTTTGATATATATATTTTTATTTTTTA
                TATATATCAATATATGTAC
SEQ ID NO: 161  GTACATATATTTTTTTATTTTTGATATATGTATTTTTATTTTTTA
                CATATATCAATATATGTAC
SEQ ID NO: 162  GTACATATATTTTTTTATTTTTGATCATGTATTTTTTATTTTTAT
                ACATGATCAATATATGTAC
SEQ ID NO: 163  GTACATATATTTTTTTATTTTTGATCATATATTTTTTATTTTTAT
                ATATGATCAATATATGTAC
SEQ ID NO: 164  GATGTATATACTTTTTATTTTTTATATATATATTTTTATTTTTTA
                TATATATAGTATATACATC
SEQ ID NO: 165  GGTACATATATTTTTTATTTTTGATATATATATTTTTATTTTTTA
                TATATATCATATATGTACC
SEQ ID NO: 166  GGTACATATATTTTTTATTTTTGATATATGTATTTTTATTTTTTA
                CATATATCATATATGTACC
SEQ ID NO: 167  GGTACATATATTTTTTATTTTTGATCATGTATTTTTTATTTTTAT
                ACATGATCATATATGTACC
SEQ ID NO: 168  GGTACATATATTTTTTATTTTTGATCATATATTTTTTATTTTTAT
                ATATGATCATATATGTACC
SEQ ID NO: 169  CGATCATATATTTTTTTATTTTTGATATATATATTTTTATTTTTT
                ATATATATCAATATATGATCG
SEQ ID NO: 170  CGATCATATATTTTTTTATTTTTGATATATGTATTTTTATTTTTT
                ACATATATCAATATATGATCG
SEQ ID NO: 171  CGATCATATATTTTTTTATTTTTGATCATGTATTTTTTATTTTTA
                TACATGATCAATATATGATCG
SEQ ID NO: 172  CGATCATATATTTTTTTATTTTTGATCATATATTTTTTATTTTTA
                TATATGATCAATATATGATCG
SEQ ID NO: 173  GATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA
                TATATATATATAGTATC
SEQ ID NO: 174  GACACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA
                TATATATATATAGTGTC
SEQ ID NO: 175  GATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA
                TATATATATATCGTATC
SEQ ID NO: 176  GATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA
                TATACATATATCGTATC
SEQ ID NO: 177  GATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT
                ATATACATGATCGTATC
SEQ ID NO: 178  GATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT
                ACATATATGATCGTATC
SEQ ID NO: 179  GGATCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA
                TATATATATATAGATCC
SEQ ID NO: 180  GACACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA
                TATATATATATCGTGTC
SEQ ID NO: 181  GACACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA
                TATACATATATCGTGTC
SEQ ID NO: 182  GACACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT
                ATATACATGATCGTGTC
SEQ ID NO: 183  GACACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT
                ACATATATGATCGTGTC
SEQ ID NO: 184  GGATCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA
                TATATATATATCGATCC
SEQ ID NO: 185  GGATCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA
                TATACATATATCGATCC
SEQ ID NO: 186  GGATCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT
                ATATACATGATCGATCC
SEQ ID NO: 187  GGATCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT
                ACATATATGATCGATCC
SEQ ID NO: 188  GCGTCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA
                TATATATATATAGACGC
SEQ ID NO: 189  GCGTCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA
                TATATATATATCGACGC
SEQ ID NO: 190  GCGTCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA
                TATACATATATCGACGC
SEQ ID NO: 191  GCGTCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT
                ATATACATGATCGACGC
SEQ ID NO: 192  GCGTCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT
                ACATATATGATCGACGC
SEQ ID NO: 193  GTATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT
                ATATATATATCGTATAC
SEQ ID NO: 194  GTATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTAT
                ATACATATATCGTATAC
SEQ ID NO: 195  GTATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA
                TATACATGATCGTATAC
SEQ ID NO: 196  GTATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTA
                CATATATGATCGTATAC
SEQ ID NO: 197  GTGATCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT
                ATATATATATCGATCAC
SEQ ID NO: 198  GTGATCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTAT
                ATACATATATCGATCAC
SEQ ID NO: 199  GTGATCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA
                TATACATGATCGATCAC
SEQ ID NO: 200  GTGATCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTA
                CATATATGATCGATCAC
SEQ ID NO: 201  GGATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT
                ATATATATATCGTATCC
SEQ ID NO: 202  GGATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTAT
                ATACATATATCGTATCC
SEQ ID NO: 203  GGATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA
                TATACATGATCGTATCC
SEQ ID NO: 204  GGATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTA
                CATATATGATCGTATCC
SEQ ID NO: 205  GCGATCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT
                ATATATATATCGATCGC
SEQ ID NO: 206  GCGATCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTAT
                ATACATATATCGATCGC
SEQ ID NO: 207  GCGATCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA
                TATACATGATCGATCGC
SEQ ID NO: 208  GCGATCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTA
                CATATATGATCGATCGC
SEQ ID NO: 209  GATATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA
                TATATATATAGTATATC
SEQ ID NO: 210  GATATATTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA
                TACATATATCATATATC
SEQ ID NO: 211  GATATATTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                ATACATGATCATATATC
SEQ ID NO: 212  GATATATTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                ATATATGATCATATATC
SEQ ID NO: 213  GTGATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA
                TATATATATAGTATCAC
SEQ ID NO: 214  GATATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA
                TATATATATCGTATATC
SEQ ID NO: 215  GATATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                ATACATGATCGTATATC
SEQ ID NO: 216  GATATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                ATATATGATCGTATATC
SEQ ID NO: 217  GGTATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA
                TATATATATAGTATACC
SEQ ID NO: 218  GTGATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA
                TATATATATCGTATCAC
SEQ ID NO: 219  GTGATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA
                TACATATATCGTATCAC
SEQ ID NO: 220  GTGATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                ATACATGATCGTATCAC
SEQ ID NO: 221  GTGATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                ATATATGATCGTATCAC
SEQ ID NO: 222  GGTATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA
                TATATATATCGTATACC
SEQ ID NO: 223  GGTATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA
                TACATATATCGTATACC
SEQ ID NO: 224  GGTATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                ATACATGATCGTATACC
SEQ ID NO: 225  GGTATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                ATATATGATCGTATACC
SEQ ID NO: 226  GGTGTACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA
                TATATATATAGTACACC
SEQ ID NO: 227  GGTGTACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA
                TATATATATCGTACACC
SEQ ID NO: 228  GGTGTACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA
                TACATATATCGTACACC
SEQ ID NO: 229  GGTGTACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                ATACATGATCGTACACC
SEQ ID NO: 230  GGTGTACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                ATATATGATCGTACACC
SEQ ID NO: 231  GTATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT
                ATATATATCGTATATAC
SEQ ID NO: 232  GTATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTAT
                ACATATATCGTATATAC
SEQ ID NO: 233  GTATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA
                TACATGATCGTATATAC
SEQ ID NO: 234  GTATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACA
                TATATGATCGTATATAC
SEQ ID NO: 235  GGATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT
                ATATATATCGTATATCC
SEQ ID NO: 236  GGATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTAT
                ACATATATCGTATATCC
SEQ ID NO: 237  GGATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA
                TACATGATCGTATATCC
SEQ ID NO: 238  GGATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACA
                TATATGATCGTATATCC
SEQ ID NO: 239  GGTGATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT
                ATATATATCGTATCACC
SEQ ID NO: 240  GGTGATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTAT
                ACATATATCGTATCACC
SEQ ID NO: 241  GGTGATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA
                TACATGATCGTATCACC
SEQ ID NO: 242  GGTGATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACA
                TATATGATCGTATCACC
SEQ ID NO: 243  GGTGATCCTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT
                ATATATATCGGATCACC
SEQ ID NO: 244  GGTGATCCTTTTTATTTTTGATAAATGTATATTTTTATTTTTTAT
                ACATATATCGGATCACC
SEQ ID NO: 245  GGTGATCCTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA
                TACATGATCGGATCACC
SEQ ID NO: 246  GGTGATCCTTTTTATTTTTGATGATATATGTTTTTTATTTTTACA
                TATATGATCGGATCACC
SEQ ID NO: 247  GTATATACATTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                ATATATCATGTATATAC
SEQ ID NO: 248  GTATATACATTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                ATATATCATGTATATAC
SEQ ID NO: 249  GTATATACATTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                CATGATCATGTATATAC
SEQ ID NO: 250  GTATATACATTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                TATGATCATGTATATAC
SEQ ID NO: 251  GGATATACATTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                ATATATCATGTATATCC
SEQ ID NO: 252  GGATATACATTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                ATATATCATGTATATCC
SEQ ID NO: 253  GGATATACATTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                CATGATCATGTATATCC
SEQ ID NO: 254  GGATATACATTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                TATGATCATGTATATCC
SEQ ID NO: 255  GGATATACACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                ATATATCGTGTATATCC
SEQ ID NO: 256  GGATATACACTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                ATATATCGTGTATATCC
SEQ ID NO: 257  GGATATACACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                CATGATCGTGTATATCC
SEQ ID NO: 258  GGATATACACTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                TATGATCGTGTATATCC
SEQ ID NO: 259  GGGTATATACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                ATATATCGTATATACCC
SEQ ID NO: 260  GGGTATATACTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                ATATATCGTATATACCC
SEQ ID NO: 261  GGGTATATACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                CATGATCGTATATACCC
SEQ ID NO: 262  GGGTATATACTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                TATGATCGTATATACCC
SEQ ID NO: 263  GGATGTACACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                ATATATCGTGTACATCC
SEQ ID NO: 264  GGATGTACACTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                ATATATCGTGTACATCC
SEQ ID NO: 265  GGATGTACACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                CATGATCGTGTACATCC
SEQ ID NO: 266  GGATGTACACTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                TATGATCGTGTACATCC
SEQ ID NO: 267  GTATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTT
                ATATATATATATATAGTATATAC
SEQ ID NO: 268  GTATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTT
                ATATATATATATATCGTATATAC
SEQ ID NO: 269  GTATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT
                ATATATACATATATCGTATATAC
SEQ ID NO: 270  GTATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTT
                TATATATACATGATCGTATATAC
SEQ ID NO: 271  GTATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT
                AGTACATATATGATCGTATATAC
SEQ ID NO: 272  GGATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTT
                ATATATATATATATAGTATATCC
SEQ ID NO: 273  GGATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTT
                ATATATATATATATCGTATATCC
SEQ ID NO: 274  GGATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT
                ATATATACATATATCGTATATCC
SEQ ID NO: 275  GGATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTT
                TATATATACATGATCGTATATCC
SEQ ID NO: 276  GGATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT
                AGTACATATATGATCGTATATCC
SEQ ID NO: 277  GGTGATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTT
                ATATATATATATATAGTATCACC
SEQ ID NO: 278  GGTGATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTT
                ATATATATATATATCGTATCACC
SEQ ID NO: 279  GGTGATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT
                ATATATACATATATCGTATCACC
SEQ ID NO: 280  GGTGATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTT
                TATATATACATGATCGTATCACC
SEQ ID NO: 281  GGTGATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT
                AGTACATATATGATCGTATCACC
SEQ ID NO: 282  GGTGATCCTTTTTATTTTTTATAAATATATATATTTTTTATTTTT
                ATATATATATATATAGGATCACC
SEQ ID NO: 283  GGTGATCCTTTTTATTTTTGATAAATATATATATTTTTTATTTTT
                ATATATATATATATCGGATCACC
SEQ ID NO: 284  GGTGATCCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT
                ATATATACATATATCGGATCACC
SEQ ID NO: 285  GGTGATCCTTTTTATTTTTGATGATGTATATATATTTTTATTTTT
                TATATATACATGATCGGATCACC
SEQ ID NO: 286  GGTGATCCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT
                AGTACATATATGATCGGATCACC
SEQ ID NO: 287  GATATATCACTTTTTATTTTTTATAAATATATATATTTTTTATTT
                TTATATATATATATATAGTGATATATC
SEQ ID NO: 288  GTATATACATTTTTTATTTTTGATAAATATATATATTTTTTATTT
                TTATATATATATATATCATGTATATAC
SEQ ID NO: 289  GTATATACATTTTTTATTTTTGATAAATGTATATATTTTTTATTT
                TTATATATACATATATCATGTATATAC
SEQ ID NO: 290  GTATATACATTTTTTATTTTTGATGATGTATATATATTTTTATTT
                TTTATATATACATGATCATGTATATAC
SEQ ID NO: 291  GTATATACATTTTTTATTTTTGATGATATATGTACTTTTTTATTT
                TTAGTACATATATGATCATGTATATAC
SEQ ID NO: 292  GGATATACACTTTTTATTTTTTATAAATATATATATTTTTTATTT
                TTATATATATATATATAGTGTATATCC
SEQ ID NO: 293  GGATATACATTTTTTATTTTTGATAAATATATATATTTTTTATTT
                TTATATATATATATATCATGTATATCC
SEQ ID NO: 294  GGATATACATTTTTTATTTTTGATAAATGTATATATTTTTTATTT
                TTATATATACATATATCATGTATATCC
SEQ ID NO: 295  GGATATACATTTTTTATTTTTGATGATGTATATATATTTTTATTT
                TTTATATATACATGATCATGTATATCC
SEQ ID NO: 296  GGATATACATTTTTTATTTTTGATGATATATGTACTTTTTTATTT
                TTAGTACATATATGATCATGTATATCC
SEQ ID NO: 297  GGGTATATACTTTTTATTTTTTATAAATATATATATTTTTTATTT
                TTATATATATATATATAGTATATACCC
SEQ ID NO: 298  GGATATACACTTTTTATTTTTGATAAATATATATATTTTTTATT
                TTTATATATATATATATCGTGTATATCC
SEQ ID NO: 299  GGATATACACTTTTTATTTTTGATAAATGTATATATTTTTTATT
                TTTATATATACATATATCGTGTATATCC
SEQ ID NO: 300  GGATATACACTTTTTATTTTTGATGATGTATATATATTTTTATT
                TTTTATATATACATGATCGTGTATATCC
SEQ ID NO: 301  GGATATACACTTTTTATTTTTGATGATATATGTACTTTTTTATTT
                TTAGTACATATATGATCGTGTATATCC
SEQ ID NO: 302  GGGTATATACTTTTTATTTTTGATAAATATATATATTTTTTATTT
                TTATATATATATATATCGTATATACCC
SEQ ID NO: 303  GGGTATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTT
                TTATATATACATATATCGTATATACCC
SEQ ID NO: 304  GGGTATATACTTTTTATTTTTGATGATGTATATATATTTTTATTT
                TTTATATATACATGATCGTATATACCC
SEQ ID NO: 305  GGGTATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTT
                TTAGTACATATATGATCGTATATACCC
SEQ ID NO: 306  GTATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT
                ATATATATATATCGTATATAC
SEQ ID NO: 307  GTATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTT
                ATATACATATATCGTATATAC
SEQ ID NO: 308  GTATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA
                TATATACATGATCGTATATAC
SEQ ID NO: 309  GTATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTG
                TACATATATGATCGTATATAC
SEQ ID NO: 310  GGATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT
                ATATATATATATCGTATATCC
SEQ ID NO: 311  GGATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTT
                ATATACATATATCGTATATCC
SEQ ID NO: 312  GGATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA
                TATATACATGATCGTATATCC
SEQ ID NO: 313  GGATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTG
                TACATATATGATCGTATATCC
SEQ ID NO: 314  GGTGATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT
                ATATATATATATCGTATCACC
SEQ ID NO: 315  GGTGATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTT
                ATATACATATATCGTATCACC
SEQ ID NO: 316  GGTGATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA
                TATATACATGATCGTATCACC
SEQ ID NO: 317  GGTGATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTG
                TACATATATGATCGTATCACC
SEQ ID NO: 318  GGTGATCCTTTTTATTTTTGATAAATATATATATTTTTATTTTTT
                ATATATATATATCGGATCACC
SEQ ID NO: 319  GGTGATCCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTT
                ATATACATATATCGGATCACC
SEQ ID NO: 320  GGTGATCCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA
                TATATACATGATCGGATCACC
SEQ ID NO: 321  GGTGATCCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTG
                TACATATATGATCGGATCACC
SEQ ID NO: 322  GTATATACATTTTTTATTTTTGATAAATATATATATTTTTATTTT
                TTATATATATATATCATGTATATAC
SEQ ID NO: 323  GTATATACATTTTTTATTTTTGATGATGTATATATTTTTTATTTT
                TATATATACATGATCATGTATATAC
SEQ ID NO: 324  GTATATACATTTTTTATTTTTGATGATATATGTACTTTTTATTTT
                TGTACATATATGATCATGTATATAC
SEQ ID NO: 325  GGATATACATTTTTTATTTTTGATAAATATATATATTTTTATTTT
                TTATATATATATATCATGTATATCC
SEQ ID NO: 326  GGATATACATTTTTTATTTTTGATAAATGTATATATTTTTATTTT
                TTATATACATATATCATGTATATCC
SEQ ID NO: 327  GGATATACATTTTTTATTTTTGATGATGTATATATTTTTTATTTT
                TATATATACATGATCATGTATATCC
SEQ ID NO: 328  GGATATACATTTTTTATTTTTGATGATATATGTACTTTTTATTTT
                TGTACATATATGATCATGTATATCC
SEQ ID NO: 329  GGATATACACTTTTTATTTTTGATAAATATATATATTTTTATTT
                TTTATATATATATATCGTGTATATCC
SEQ ID NO: 330  GGATATACACTTTTTATTTTTGATAAATGTATATATTTTTATTT
                TTTATATACATATATCGTGTATATCC
SEQ ID NO: 331  GGATATACACTTTTTATTTTTGATGATGTATATATTTTTTATTTT
                TATATATACATGATCGTGTATATCC
SEQ ID NO: 332  GGATATACACTTTTTATTTTTGATGATATATGTACTTTTTATTTT
                TGTACATATATGATCGTGTATATCC
SEQ ID NO: 333  GGGTATATACTTTTTATTTTTGATAAATATATATATTTTTATTTT
                TTATATATATATATCGTATATACCC
SEQ ID NO: 334  GGGTATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTT
                TTATATACATATATCGTATATACCC
SEQ ID NO: 335  GGGTATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTT
                TATATATACATGATCGTATATACCC
SEQ ID NO: 336  GGGTATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTT
                TGTACATATATGATCGTATATACCC
SEQ ID NO: 337  GATATATCACTTTTTATTTTTTATAAATATATATTTTTTATTTTT
                ATATATATATATAGTGATATATC
SEQ ID NO: 338  GTATATACATTTTTTATTTTTGATAAATATATATTTTTTATTTTT
                ATATATATATATCATGTATATAC
SEQ ID NO: 339  GTATATACATTTTTTATTTTTGATGATGTATATATTTTTATTTTT
                TATATACATGATCATGTATATAC
SEQ ID NO: 340  GTATATACATTTTTTATTTTTGATGATATATGTATTTTTATTTTT
                TACATATATGATCATGTATATAC
SEQ ID NO: 341  GGATATACACTTTTTATTTTTTATAAATATATATTTTTTATTTTT
                ATATATATATATAGTGTATATCC
SEQ ID NO: 342  GGATATACATTTTTTATTTTTGATAAATATATATTTTTTATTTTT
                ATATATATATATCATGTATATCC
SEQ ID NO: 343  GGATATACATTTTTTATTTTTGATAAATGTATATTTTTTATTTTT
                ATATACATATATCATGTATATCC
SEQ ID NO: 344  GGATATACATTTTTTATTTTTGATGATGTATATATTTTTATTTTT
                TATATACATGATCATGTATATCC
SEQ ID NO: 345  GGATATACATTTTTTATTTTTGATGATATATGTATTTTTATTTTT
                TACATATATGATCATGTATATCC
SEQ ID NO: 346  GGGTATATACTTTTTATTTTTTATAAATATATATTTTTTATTTTT
                ATATATATATATAGTATATACCC
SEQ ID NO: 347  GGATATACACTTTTTATTTTTGATAAATATATATTTTTTATTTTT
                ATATATATATATCGTGTATATCC
SEQ ID NO: 348  GGATATACACTTTTTATTTTTGATAAATGTATATTTTTTATTTTT
                ATATACATATATCGTGTATATCC
SEQ ID NO: 349  GGATATACACTTTTTATTTTTGATGATGTATATATTTTTATTTTT
                TATATACATGATCGTGTATATCC
SEQ ID NO: 350  GGATATACACTTTTTATTTTTGATGATATATGTATTTTTATTTTT
                TACATATATGATCGTGTATATCC
SEQ ID NO: 351  GGGTATATACTTTTTATTTTTGATAAATATATATTTTTTATTTTT
                ATATATATATATCGTATATACCC
SEQ ID NO: 352  GGGTATATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTT
                ATATACATATATCGTATATACCC
SEQ ID NO: 353  GGGTATATACTTTTTATTTTTGATGATGTATATATTTTTATTTTT
                TATATACATGATCGTATATACCC
SEQ ID NO: 354  GGGTATATACTTTTTATTTTTGATGATATATGTATTTTTATTTTT
                TACATATATGATCGTATATACCC
SEQ ID NO: 355  GTATATACATTTTTTATTTTTGATAAATATATATTTTTATTTTTT
                ATATATATATCATGTATATAC
SEQ ID NO: 356  GTATATACATTTTTTATTTTTGATGATGTATATTTTTTATTTTTA
                TATACATGATCATGTATATAC
SEQ ID NO: 357  GTATATACATTTTTTATTTTTGATGATATATGTTTTTTATTTTTA
                CATATATGATCATGTATATAC
SEQ ID NO: 358  GGATATACATTTTTTATTTTTGATAAATATATATTTTTATTTTTT
                ATATATATATCATGTATATCC
SEQ ID NO: 359  GGATATACATTTTTTATTTTTGATAAATGTATATTTTTATTTTTT
                ATACATATATCATGTATATCC
SEQ ID NO: 360  GGATATACATTTTTTATTTTTGATGATGTATATTTTTTATTTTTA
                TATACATGATCATGTATATCC
SEQ ID NO: 361  GGATATACATTTTTTATTTTTGATGATATATGTTTTTTATTTTTA
                CATATATGATCATGTATATCC
SEQ ID NO: 362  GGATATACACTTTTTATTTTTGATAAATATATATTTTTATTTTTT
                ATATATATATCGTGTATATCC
SEQ ID NO: 363  GGATATACACTTTTTATTTTTGATAAATGTATATTTTTATTTTTT
                ATACATATATCGTGTATATCC
SEQ ID NO: 364  GGATATACACTTTTTATTTTTGATGATGTATATTTTTTATTTTTA
                TATACATGATCGTGTATATCC
SEQ ID NO: 365  GGATATACACTTTTTATTTTTGATGATATATGTTTTTTATTTTTA
                CATATATGATCGTGTATATCC
SEQ ID NO: 366  GGGTATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTT
                ATATATATATCGTATATACCC
SEQ ID NO: 367  GGGTATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTT
                ATACATATATCGTATATACCC
SEQ ID NO: 368  GGGTATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTA
                TATACATGATCGTATATACCC
SEQ ID NO: 369  GGGTATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTA
                CATATATGATCGTATATACCC
SEQ ID NO: 370  GTACATATATTTTTTTATTTTTGATAAATATATTTTTATTTTTTA
                TATATATCAATATATGTAC
SEQ ID NO: 371  GTACATATATTTTTTTATTTTTGATAAATGTATTTTTATTTTTTA
                CATATATCAATATATGTAC
SEQ ID NO: 372  GTACATATATTTTTTTATTTTTGATGATGTATTTTTTATTTTTAT
                ACATGATCAATATATGTAC
SEQ ID NO: 373  GTACATATATTTTTTTATTTTTGATGATATATTTTTTATTTTTAT
                ATATGATCAATATATGTAC
SEQ ID NO: 374  GGTACATATATTTTTTATTTTTGATAAATATATTTTTATTTTTTA
                TATATATCATATATGTACC
SEQ ID NO: 375  GGTACATATATTTTTTATTTTTGATAAATGTATTTTTATTTTTTA
                CATATATCATATATGTACC
SEQ ID NO: 376  GGTACATATATTTTTTATTTTTGATGATGTATTTTTTATTTTTAT
                ACATGATCATATATGTACC
SEQ ID NO: 377  GGTACATATATTTTTTATTTTTGATGATATATTTTTTATTTTTAT
                ATATGATCATATATGTACC
SEQ ID NO: 378  CGATCATATATTTTTTTATTTTTGATAAATATATTTTTATTTTTT
                ATATATATCAATATATGATCG
SEQ ID NO: 379  CGATCATATATTTTTTTATTTTTGATAAATGTATTTTTATTTTTT
                ACATATATCAATATATGATCG
SEQ ID NO: 380  CGATCATATATTTTTTTATTTTTGATGATGTATTTTTTATTTTTA
                TACATGATCAATATATGATCG
SEQ ID NO: 381  CGATCATATATTTTTTTATTTTTGATGATATATTTTTTATTTTTA
                TATATGATCAATATATGATCG
SEQ ID NO: 382  GTATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT
                TATATATACATGATCGTATATAC
SEQ ID NO: 383  GTATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTT
                AGTACATATATGATCGTATATAC
SEQ ID NO: 384  GGATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT
                TATATATACATGATCGTATATCC
SEQ ID NO: 385  GGATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTT
                AGTACATATATGATCGTATATCC
SEQ ID NO: 386  GGTGATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT
                TATATATACATGATCGTATCACC
SEQ ID NO: 387  GGTGATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTT
                AGTACATATATGATCGTATCACC
SEQ ID NO: 388  GGTGATCCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT
                TATATATACATGATCGGATCACC
SEQ ID NO: 389  GGTGATCCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTT
                AGTACATATATGATCGGATCACC
SEQ ID NO: 390  GTATATACATTTTTTATTTTTGATGATGTAAATATATTTTTATTT
                TTTATATATACATGATCATGTATATAC
SEQ ID NO: 391  GTATATACATTTTTTATTTTTGATGATATAAGTACTTTTTTATTT
                TTAGTACATATATGATCATGTATATAC
SEQ ID NO: 392  GGATATACATTTTTTATTTTTGATAAATGTAAATATTTTTTATT
                TTTATATATACATATATCATGTATATCC
SEQ ID NO: 393  GGATATACATTTTTTATTTTTGATGATGTAAATATATTTTTATT
                TTTTATATATACATGATCATGTATATCC
SEQ ID NO: 394  GGATATACATTTTTTATTTTTGATGATATAAGTACTTTTTTATT
                TTTAGTACATATATGATCATGTATATCC
SEQ ID NO: 395  GGATATACACTTTTTATTTTTGATGATGTAAATATATTTTTATT
                TTTTATATATACATGATCGTGTATATCC
SEQ ID NO: 396  GGATATACACTTTTTATTTTTGATGATATAAGTACTTTTTTATT
                TTTAGTACATATATGATCGTGTATATCC
SEQ ID NO: 397  GGGTATATACTTTTTATTTTTGATGATGTAAATATATTTTTATT
                TTTTATATATACATGATCGTATATACCC
SEQ ID NO: 398  GGGTATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATT
                TTTAGTACATATATGATCGTATATACCC
SEQ ID NO: 399  GTATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA
                TATATACATGATCGTATATAC
SEQ ID NO: 400  GTATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTG
                TACATATATGATCGTATATAC
SEQ ID NO: 401  GGATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA
                TATATACATGATCGTATATCC
SEQ ID NO: 402  GGATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTT
                GTACATATATGATCGTATATCC
SEQ ID NO: 403  GGTGATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA
                TATATACATGATCGTATCACC
SEQ ID NO: 404  GGTGATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTT
                GTACATATATGATCGTATCACC
SEQ ID NO: 405  GGTGATCCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA
                TATATACATGATCGGATCACC
SEQ ID NO: 406  GGTGATCCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTG
                TACATATATGATCGGATCACC
SEQ ID NO: 407  GTATATACATTTTTTATTTTTGATGATGTAAATATTTTTTATTTT
                TATATATACATGATCATGTATATAC
SEQ ID NO: 408  GTATATACATTTTTTATTTTTGATGATATAAGTACTTTTTATTTT
                TGTACATATATGATCATGTATATAC
SEQ ID NO: 409  GGATATACATTTTTTATTTTTGATGATGTAAATATTTTTTATTTT
                TATATATACATGATCATGTATATCC
SEQ ID NO: 410  GGATATACATTTTTTATTTTTGATGATATAAGTACTTTTTATTT
                TTGTACATATATGATCATGTATATCC
SEQ ID NO: 411  GGATATACACTTTTTATTTTTGATGATGTAAATATTTTTTATTT
                TTATATATACATGATCGTGTATATCC
SEQ ID NO: 412  GGATATACACTTTTTATTTTTGATGATATAAGTACTTTTTATTT
                TTGTACATATATGATCGTGTATATCC
SEQ ID NO: 413  GGGTATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTT
                TATATATACATGATCGTATATACCC
SEQ ID NO: 414  GGGTATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTT
                TTGTACATATATGATCGTATATACCC
SEQ ID NO: 415  GTATATACATTTTTTATTTTTGATGATATAAGTATTTTTATTTTT
                TACATATATGATCATGTATATAC
SEQ ID NO: 416  GGATATACATTTTTTATTTTTGATAAATGAATATTTTTTATTTTT
                ATATACATATATCATGTATATCC
SEQ ID NO: 417  GGATATACATTTTTTATTTTTGATGATATAAGTATTTTTATTTTT
                TACATATATGATCATGTATATCC
SEQ ID NO: 418  GGATATACACTTTTTATTTTTGATAAATGAATATTTTTTATTTT
                TATATACATATATCGTGTATATCC
SEQ ID NO: 419  GGATATACACTTTTTATTTTTGATGATATAAGTATTTTTATTTT
                TTACATATATGATCGTGTATATCC
SEQ ID NO: 420  GGGTATATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTT
                ATATACATATATCGTATATACCC
SEQ ID NO: 421  GGGTATATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTT
                TACATATATGATCGTATATACCC
SEQ ID NO: 422  GTATATACATTTTTTATTTTTGATGATGAATATTTTTTATTTTTA
                TATACATGATCATGTATATAC
SEQ ID NO: 423  GGATATACATTTTTTATTTTTGATAAATGAATATTTTTATTTTTT
                ATACATATATCATGTATATCC
SEQ ID NO: 424  GGATATACATTTTTTATTTTTGATGATGAATATTTTTTATTTTTA
                TATACATGATCATGTATATCC
SEQ ID NO: 425  GGATATACATTTTTTATTTTTGATGATAAATGTTTTTTATTTTTA
                CATATATGATCATGTATATCC
SEQ ID NO: 426  GGATATACACTTTTTATTTTTGATGATGAATATTTTTTATTTTT
                ATATACATGATCGTGTATATCC
SEQ ID NO: 427  GGGTATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTA
                TATACATGATCGTATATACCC
SEQ ID NO: 428  GATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTAT
                ATATACATGATCGTATC
SEQ ID NO: 429  GATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT
                ACATATATGATCGTATC
SEQ ID NO: 430  GACACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTAT
                ATATACATGATCGTGTC
SEQ ID NO: 431  GACACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT
                ACATATATGATCGTGTC
SEQ ID NO: 432  GGATCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTAT
                ATATACATGATCGATCC
SEQ ID NO: 433  GGATCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT
                ACATATATGATCGATCC
SEQ ID NO: 434  GCGTCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTAT
                ATATACATGATCGACGC
SEQ ID NO: 435  GCGTCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT
                ACATATATGATCGACGC
SEQ ID NO: 436  GTATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATA
                TATACATGATCGTATAC
SEQ ID NO: 437  GTATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA
                CATATATGATCGTATAC
SEQ ID NO: 438  GTGATCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATA
                TATACATGATCGATCAC
SEQ ID NO: 439  GTGATCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA
                CATATATGATCGATCAC
SEQ ID NO: 440  GGATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATA
                TATACATGATCGTATCC
SEQ ID NO: 441  GGATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGT
                ACATATATGATCGTATCC
SEQ ID NO: 442  GCGATCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATA
                TATACATGATCGATCGC
SEQ ID NO: 443  GCGATCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA
                CATATATGATCGATCGC
SEQ ID NO: 444  GATATATTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                ATATATGATCATATATC
SEQ ID NO: 445  GATATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                ATATATGATCGTATATC
SEQ ID NO: 446  GTGATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATA
                TACATATATCGTATCAC
SEQ ID NO: 447  GTGATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                ATATATGATCGTATCAC
SEQ ID NO: 448  GGTATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATA
                TACATATATCGTATACC
SEQ ID NO: 449  GGTATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                ATATATGATCGTATACC
SEQ ID NO: 450  GGTGTACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATA
                TACATATATCGTACACC
SEQ ID NO: 451  GGTGTACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                ATATATGATCGTACACC
SEQ ID NO: 452  GTATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA
                TACATGATCGTATATAC
SEQ ID NO: 453  GTATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACA
                TATATGATCGTATATAC
SEQ ID NO: 454  GGATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA
                TACATGATCGTATATCC
SEQ ID NO: 455  GGTGATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA
                TACATGATCGTATCACC
SEQ ID NO: 456  GGTGATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTAC
                ATATATGATCGTATCACC
SEQ ID NO: 457  GGTGATCCTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA
                TACATGATCGGATCACC
SEQ ID NO: 458  GATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATA
                TATATATATATCGTATC
SEQ ID NO: 459  GATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATA
                TATACATATATCGTATC
SEQ ID NO: 460  GACACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTAT
                ATATATATATATCGTGTC
SEQ ID NO: 461  GACACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTAT
                ATATACATATATCGTGTC
SEQ ID NO: 462  GACACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTAT
                ATATACATATATCGTGTC
SEQ ID NO: 463  GGATCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATA
                TATATATATATCGATCC
SEQ ID NO: 464  GGATCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATA
                TATACATATATCGATCC
SEQ ID NO: 465  GGATCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATA
                TATACATATATCGATCC
SEQ ID NO: 466  GCGTCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATA
                TATATATATATCGACGC
SEQ ID NO: 467  GCGTCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATA
                TATACATATATCGACGC
SEQ ID NO: 468  GCGTCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATA
                TATACATATATCGACGC
SEQ ID NO: 469  GTATATACATTTTTTATTTTTGATATAAATATATAATTTTTATTT
                TTATATATATATATATCATGTATATAC
SEQ ID NO: 470  GTATATACATTTTTTATTTTTGATAAATGAATATATTTTTTATTT
                TTATATATACATATATCATGTATATAC
SEQ ID NO: 471  GTATATACATTTTTTATTTTTGATATAAGTAAATATTTTTTATTT
                TTATATATACATATATCATGTATATAC
SEQ ID NO: 472  GGATATACATTTTTTATTTTTGATATAAATATATAATTTTTATT
                TTTATATATATATATATCATGTATATCC
SEQ ID NO: 473  GGATATACACTTTTTATTTTTGATATAAATATATAATTTTTATT
                TTTATATATATATATATCGTGTATATCC
SEQ ID NO: 474  GGATATACACTTTTTATTTTTGATAAATGAATATATTTTTTATT
                TTTATATATACATATATCGTGTATATCC
SEQ ID NO: 475  GGATATACACTTTTTATTTTTGATATAAGTAAATATTTTTTATT
                TTTATATATACATATATCGTGTATATCC
SEQ ID NO: 476  GGGTATATACTTTTTATTTTTGATATAAATATATAATTTTTATT
                TTTATATATATATATATCGTATATACCC
SEQ ID NO: 477  GGGTATATACTTTTTATTTTTGATAAATGAATATATTTTTTATT
                TTTATATATACATATATCGTATATACCC
SEQ ID NO: 478  GGGTATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATT
                TTTATATATACATATATCGTATATACCC
SEQ ID NO: 479  GTATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT
                ATATATATATAGTATAC
SEQ ID NO: 480  GTGATCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT
                ATATATATATAGATCAC
SEQ ID NO: 481  GTATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT
                ATATATATATCGTATAC
SEQ ID NO: 482  GTATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT
                ATATATATATCGTATAC
SEQ ID NO: 483  GTATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT
                ATACATATATCGTATAC
SEQ ID NO: 484  GGATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT
                ATATATATATAGTATCC
SEQ ID NO: 485  GTGATCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT
                ATATATATATCGATCAC
SEQ ID NO: 486  GTGATCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT
                ATATATATATCGATCAC
SEQ ID NO: 487  GTGATCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT
                ATACATATATCGATCAC
SEQ ID NO: 488  GGATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT
                ATATATATATCGTATCC
SEQ ID NO: 489  GGATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT
                ATATATATATCGTATCC
SEQ ID NO: 490  GGATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT
                ATACATATATCGTATCC
SEQ ID NO: 491  GCGATCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT
                ATATATATATAGATCGC
SEQ ID NO: 492  GCGATCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT
                ATATATATATCGATCGC
SEQ ID NO: 493  GCGATCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT
                ATATATATATCGATCGC
SEQ ID NO: 494  GCGATCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT
                ATACATATATCGATCGC
SEQ ID NO: 495  GATATATCACTTTTTATTTTTTATAAATATATATTTTTTTATTTT
                TTATATATATATATAGTGATATATC
SEQ ID NO: 496  GTATATACATTTTTTATTTTTGATATAAATATATTTTTTTATTTT
                TTATATATATATATCATGTATATAC
SEQ ID NO: 497  GTATATACATTTTTTATTTTTGATATATAAATATTTTTTTATTTT
                TTATATATATATATCATGTATATAC
SEQ ID NO: 498  GGATATACACTTTTTATTTTTTATAAATATATATTTTTTTATTTT
                TTATATATATATATAGTGTATATCC
SEQ ID NO: 499  GGATATACATTTTTTATTTTTGATATAAATATATTTTTTTATTTT
                TTATATATATATATCATGTATATCC
SEQ ID NO: 500  GGATATACATTTTTTATTTTTGATATATAAATATTTTTTTATTTT
                TTATATATATATATCATGTATATCC
SEQ ID NO: 501  GGATATACATTTTTTATTTTTGATAAATGAATATATTTTTATTT
                TTTATATACATATATCATGTATATCC
SEQ ID NO: 502  GGGTATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTT
                TTATATATATATATAGTATATACCC
SEQ ID NO: 503  GGATATACACTTTTTATTTTTGATATAAATATATTTTTTTATTTT
                TTATATATATATATCGTGTATATCC
SEQ ID NO: 504  GGATATACACTTTTTATTTTTGATATATAAATATTTTTTTATTTT
                TTATATATATATATCGTGTATATCC
SEQ ID NO: 505  GGATATACACTTTTTATTTTTGATAAATGAATATATTTTTATTT
                TTTATATACATATATCGTGTATATCC
SEQ ID NO: 506  GGGTATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTT
                TTATATATATATATCGTATATACCC
SEQ ID NO: 507  GGGTATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTT
                TTATATATATATATCGTATATACCC
SEQ ID NO: 508  GGGTATATACTTTTTATTTTTGATAAATGAATATATTTTTATTT
                TTTATATACATATATCGTATATACCC
SEQ ID NO: 509  GATATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA
                TATATATATCGTATATC
SEQ ID NO: 510  GTGATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA
                TATATATATCGTATCAC
SEQ ID NO: 511  GGTATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA
                TATATATATCGTATACC
SEQ ID NO: 512  GGTGTACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA
                TATATATATCGTACACC
SEQ ID NO: 513  GTATATACATTTTTTATTTTTGATAAATATATAATTTTTATTTTT
                ATATATATATATCATGTATATAC
SEQ ID NO: 514  GGATATACATTTTTTATTTTTGATAAATATATAATTTTTATTTTT
                ATATATATATATCATGTATATCC
SEQ ID NO: 515  GGATATACACTTTTTATTTTTGATAAATATATAATTTTTATTTT
                TATATATATATATCGTGTATATCC
SEQ ID NO: 516  GGGTATATACTTTTTATTTTTGATAAATATATAATTTTTATTTTT
                ATATATATATATCGTATATACCC
SEQ ID NO: 517  GTATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT
                ATATATATCGTATATAC
SEQ ID NO: 518  GTATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT
                ACATATATCGTATATAC
SEQ ID NO: 519  GGATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT
                ATATATATCGTATATCC
SEQ ID NO: 520  GGATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT
                ACATATATCGTATATCC
SEQ ID NO: 521  GGATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTAC
                ATATATGATCGTATATCC
SEQ ID NO: 522  GGTGATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT
                ATATATATCGTATCACC
SEQ ID NO: 523  GGTGATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT
                ACATATATCGTATCACC
SEQ ID NO: 524  GGTGATCCTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT
                ATATATATCGGATCACC
SEQ ID NO: 525  GGTGATCCTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT
                ACATATATCGGATCACC
SEQ ID NO: 526  GGTGATCCTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACA
                TATATGATCGGATCACC
SEQ ID NO: 527  GTATATACATTTTTTATTTTTGATAAATATATTTTTTTATTTTTT
                ATATATATATCATGTATATAC
SEQ ID NO: 528  GTATATACATTTTTTATTTTTGATAAATGTATTTTTTTATTTTTT
                ATACATATATCATGTATATAC
SEQ ID NO: 529  GTATATACATTTTTTATTTTTGATGATAAATGTTTTTTATTTTTA
                CATATATGATCATGTATATAC
SEQ ID NO: 530  GGATATACATTTTTTATTTTTGATAAATATATTTTTTTATTTTTT
                ATATATATATCATGTATATCC
SEQ ID NO: 531  GGATATACACTTTTTATTTTTGATAAATATATTTTTTTATTTTTT
                ATATATATATCGTGTATATCC
SEQ ID NO: 532  GGATATACACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTT
                ATACATATATCGTGTATATCC
SEQ ID NO: 533  GGATATACACTTTTTATTTTTGATGATAAATGTTTTTTATTTTT
                ACATATATGATCGTGTATATCC
SEQ ID NO: 534  GGGTATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTT
                ATATATATATCGTATATACCC
SEQ ID NO: 535  GGGTATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTT
                ATACATATATCGTATATACCC
SEQ ID NO: 536  GGGTATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTA
                CATATATGATCGTATATACCC
SEQ ID NO: 537  GTATATACATTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                ATATATCATGTATATAC
SEQ ID NO: 538  GTATATACATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                ATATATCATGTATATAC
SEQ ID NO: 539  GGATATACATTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                ATATATCATGTATATCC
SEQ ID NO: 540  GGATATACATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                ATATATCATGTATATCC
SEQ ID NO: 541  GGATATACATTTTTTATTTTTGATGATGAATTTTTTATTTTTATA
                CATGATCATGTATATCC
SEQ ID NO: 542  GGATATACACTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                ATATATCGTGTATATCC
SEQ ID NO: 543  GGATATACACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                ATATATCGTGTATATCC
SEQ ID NO: 544  GGGTATATACTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                ATATATCGTATATACCC
SEQ ID NO: 545  GGGTATATACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                ATATATCGTATATACCC
SEQ ID NO: 546  GGATGTACACTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                ATATATCGTGTACATCC
SEQ ID NO: 547  GGATGTACACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                ATATATCGTGTACATCC
SEQ ID NO: 548  GTACATATATTTTTTTATTTTTGATAAATATTTTTTTATTTTTTA
                TATATATCAATATATGTAC
SEQ ID NO: 549  GTACATATATTTTTTTATTTTTGATAAATGTTTTTTTATTTTTTA
                CATATATCAATATATGTAC
SEQ ID NO: 550  GGTACATATATTTTTTATTTTTGATAAATATTTTTTTATTTTTTA
                TATATATCATATATGTACC
SEQ ID NO: 551  GGTACATATATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTA
                CATATATCATATATGTACC
SEQ ID NO: 552  CGATCATATATTTTTTTATTTTTGATAAATATTTTTTTATTTTTT
                ATATATATCAATATATGATCG
SEQ ID NO: 553  CGATCATATATTTTTTTATTTTTGATAAATGTTTTTTTATTTTTT
                ACATATATCAATATATGATCG
SEQ ID NO: 554  CGATCATATATTTTTTTATTTTTGATGATGAATTTTTTATTTTTA
                TACATGATCAATATATGATCG
SEQ ID NO: 555  CGATCATATATTTTTTTATTTTTGATGATAAATTTTTTATTTTTA
                TATATGATCAATATATGATCG
SEQ ID NO: 556  GTATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTT
                ATATATATATATATCGTATATAC
SEQ ID NO: 557  GTATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT
                ATATATACATATATCGTATATAC
SEQ ID NO: 558  GGATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTT
                ATATATATATATATCGTATATCC
SEQ ID NO: 559  GGATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT
                ATATATACATATATCGTATATCC
SEQ ID NO: 560  GGTGATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTT
                ATATATATATATATCGTATCACC
SEQ ID NO: 561  GGTGATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT
                ATATATACATATATCGTATCACC
SEQ ID NO: 562  GGTGATCCTTTTTATTTTTGATATAAATATATAATTTTTATTTTT
                ATATATATATATATCGGATCACC
SEQ ID NO: 563  GGTGATCCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT
                ATATATACATATATCGGATCACC
SEQ ID NO: 564  GTATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT
                ATATATACATATATCGTATATAC
SEQ ID NO: 565  GGATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT
                ATATATACATATATCGTATATCC
SEQ ID NO: 566  GGTGATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT
                ATATATACATATATCGTATCACC
SEQ ID NO: 567  GGTGATCCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT
                ATATATACATATATCGGATCACC
SEQ ID NO: 568  GTATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT
                ATATATATATATAGTATATAC
SEQ ID NO: 569  GTATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT
                ATATATATATATCGTATATAC
SEQ ID NO: 570  GTATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT
                ATATACATATATCGTATATAC
SEQ ID NO: 571  GGATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT
                ATATATATATATAGTATATCC
SEQ ID NO: 572  GGATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT
                ATATATATATATCGTATATCC
SEQ ID NO: 573  GGATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT
                ATATACATATATCGTATATCC
SEQ ID NO: 574  GGTGATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT
                ATATATATATATAGTATCACC
SEQ ID NO: 575  GGTGATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT
                ATATATATATATCGTATCACC
SEQ ID NO: 576  GGTGATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT
                ATATACATATATCGTATCACC
SEQ ID NO: 577  GGTGATCCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT
                ATATATATATATAGGATCACC
SEQ ID NO: 578  GGTGATCCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT
                ATATATATATATCGGATCACC
SEQ ID NO: 579  GGTGATCCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT
                ATATACATATATCGGATCACC
SEQ ID NO: 580  GTATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT
                ATATATATATATCGTATATAC
SEQ ID NO: 581  GGATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT
                ATATATATATATCGTATATCC
SEQ ID NO: 582  GGTGATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT
                ATATATATATATCGTATCACC
SEQ ID NO: 583  GGTGATCCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT
                ATATATATATATCGGATCACC
SEQ ID NO: 584  GATACAAAAAAAAAAATATATATATATATATAAAAAAAAAAA
                ATATATATATATATAGTATC
SEQ ID NO: 585  GACACAAAAAAAAAAAGATATATATATATATAAAAAAAAAAA
                ATATATATATATATCGTGTC
SEQ ID NO: 586  GATATACAAAAAAAAAAATATATATATATATAAAAAAAAAAA
                ATATATATATATAGTATATC
SEQ ID NO: 587  GATATATAAAAAAAAAAAGATATATGTATATAAAAAAAAAAA
                ATATACATATATCATATATC
SEQ ID NO: 588  GATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAAA
                ATATATATATATCGTATATC
SEQ ID NO: 589  GGTATACAAAAAAAAAAATATATATATATATAAAAAAAAAAA
                ATATATATATATAGTATACC
SEQ ID NO: 590  GATATATCACAAAAAAAAAAATATATATATAAAAAAAAAAAA
                TATATATATAGTGATATATC
SEQ ID NO: 591  GTATATACATAAAAAAAAAAAGATATATGTAAAAAAAAAAAA
                TACATATATCATGTATATAC
SEQ ID NO: 592  GGATATACATAAAAAAAAAAAGATATATGTAAAAAAAAAAA
                ATACATATATCATGTATATCC
SEQ ID NO: 593  GGATATACATAAAAAAAAAAAGATCATGTATAAAAAAAAAAA
                ATACATGATCATGTATATCC
SEQ ID NO: 594  GGGTATATACAAAAAAAAAAATATATATATAAAAAAAAAAAA
                TATATATATAGTATATACCC
SEQ ID NO: 595  GTATATACAAAAAAAAAAATATATATATATATATAAAAAAAA
                AAAATATATATATATATAGTATATAC
SEQ ID NO: 596  GTATATACAAAAAAAAAAAGATATATATATATATAAAAAAAA
                AAAATATATATATATATCGTATATAC
SEQ ID NO: 597  GGATATACAAAAAAAAAAATATATATATATATATAAAAAAAA
                AAAATATATATATATATAGTATATCC
SEQ ID NO: 598  GGATATACAAAAAAAAAAAGATATATATATATATAAAAAAAA
                AAAATATATATATATATCGTATATCC
SEQ ID NO: 599  GTATATACAAAAAAAAAAATATATATATATATAAAAAAAAAA
                AATATATATATATATAGTATATAC
SEQ ID NO: 600  GTATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAA
                AATATATATATATATCGTATATAC
SEQ ID NO: 601  GGATATACAAAAAAAAAAATATATATATATATAAAAAAAAAA
                AATATATATATATATAGTATATCC
SEQ ID NO: 602  GGATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAA
                AATATATATATATATCGTATATCC
SEQ ID NO: 603  GATATATCACAAAAAAAAAAATATATATATATAAAAAAAAAA
                AATATATATATATAGTGATATATC
SEQ ID NO: 604  GGATATACATAAAAAAAAAAAGATATATATATAAAAAAAAAA
                AATATATATATATCATGTATATCC
SEQ ID NO: 605  GTACATATATTAAAAAAAAAAAGATATATATAAAAAAAAAAA
                ATATATATATCAATATATGTAC
SEQ ID NO: 606  GATGTATATACAAAAAAAAAAATATATATATAAAAAAAAAAA
                ATATATATATAGTATATACATC
SEQ ID NO: 607  CGATCATATATTAAAAAAAAAAAGATATATATAAAAAAAAAA
                AATATATATATCAATATATGATCG
SEQ ID NO: 608  CGATCATATATTAAAAAAAAAAAGATATATGTAAAAAAAAAA
                AATACATATATCAATATATGATCG
SEQ ID NO: 609  GATACAAAAAAAAAAATATAAATATATATATAAAAAAAAAAA
                ATATATATATATATAGTATC
SEQ ID NO: 610  GGATCAAAAAAAAAAATATAAATATATATATAAAAAAAAAAA
                ATATATATATATATAGATCC
SEQ ID NO: 611  GACACAAAAAAAAAAAGATAAATATATATATAAAAAAAAAA
                AATATATATATATATCGTGTC
SEQ ID NO: 612  GACACAAAAAAAAAAAGATGATGTATATATAAAAAAAAAAA
                ATATATATACATGATCGTGTC
SEQ ID NO: 613  GCGTCAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAA
                ATATATATATATATCGACGC
SEQ ID NO: 614  GATATACAAAAAAAAAAATATAAATATATATAAAAAAAAAAA
                ATATATATATATAGTATATC
SEQ ID NO: 615  GTATATACATAAAAAAAAAAAGATAAATGTAAAAAAAAAAA
                ATACATATATCATGTATATAC
SEQ ID NO: 616  GTATATACATAAAAAAAAAAAGATGATATATAAAAAAAAAAA
                ATATATGATCATGTATATAC
SEQ ID NO: 617  GGATATACATAAAAAAAAAAAGATAAATATAAAAAAAAAAA
                ATATATATATCATGTATATCC
SEQ ID NO: 618  GGATATACATAAAAAAAAAAAGATGATATATAAAAAAAAAA
                AATATATGATCATGTATATCC
SEQ ID NO: 619  GTATATACAAAAAAAAAAATATAAATATATATATAAAAAAAA
                AAAATATATATATATATAGTATATAC
SEQ ID NO: 620  GTATATACAAAAAAAAAAAGATAAATATATATATAAAAAAAA
                AAAATATATATATATATCGTATATAC
SEQ ID NO: 621  GGATATACAAAAAAAAAAATATAAATATATATATAAAAAAAA
                AAAATATATATATATATAGTATATCC
SEQ ID NO: 622  GGATATACAAAAAAAAAAAGATAAATATATATATAAAAAAAA
                AAAATATATATATATATCGTATATCC
SEQ ID NO: 623  GTATATACAAAAAAAAAAAGATAAATGTATATAAAAAAAAAA
                AATATATACATATATCGTATATAC
SEQ ID NO: 624  GGATATACAAAAAAAAAAAGATAAATGTATATAAAAAAAAA
                AAATATATACATATATCGTATATCC
SEQ ID NO: 625  GGTGATACAAAAAAAAAAAGATGATGTATATATAAAAAAAAA
                AAATATATACATGATCGTATCACC
SEQ ID NO: 626  GATATATCACAAAAAAAAAAATATAAATATATATAAAAAAAA
                AAAATATATATATATAGTGATATATC
SEQ ID NO: 627  GTATATACATAAAAAAAAAAAGATAAATATATAAAAAAAAAA
                AATATATATATATCATGTATATAC
SEQ ID NO: 628  GTATATACATAAAAAAAAAAAGATGATATATGTAAAAAAAAA
                AAACATATATGATCATGTATATAC
SEQ ID NO: 629  GGATATACATAAAAAAAAAAAGATAAATATATAAAAAAAAA
                AAATATATATATATCATGTATATCC
SEQ ID NO: 630  GTACATATATTAAAAAAAAAAAGATAAATATAAAAAAAAAAA
                ATATATATATCAATATATGTAC
SEQ ID NO: 631  GTACATATATTAAAAAAAAAAAGATAAATGTAAAAAAAAAAA
                ATACATATATCAATATATGTAC
SEQ ID NO: 632  GTACATATATTAAAAAAAAAAAGATGATATATAAAAAAAAAA
                AATATATGATCAATATATGTAC
SEQ ID NO: 633  GGATATACATAAAAAAAAAAAGATGATGAATAAAAAAAAAA
                AATACATGATCATGTATATCC
SEQ ID NO: 634  GTATATACATAAAAAAAAAAAGATAAATGTTAAAAAAAAAAA
                TACATATATCATGTATATAC
SEQ ID NO: 635  GATACAAAAAAAAAAAGATATAAATATATAAAAAAAAAAAA
                AATATATATATATATCGTATC
SEQ ID NO: 636  GATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAAAA
                AAGTACATATATGATCGTATC
SEQ ID NO: 637  GACACAAAAAAAAAAAGATAAATGAATATATAAAAAAAAAA
                AATATATACATATATCGTGTC
SEQ ID NO: 638  GGATATACAAAAAAAAAAAGATATAAGTAAATATAAAAAAA
                AAAAATATATACATATATCGTATATCC
SEQ ID NO: 639  GGATATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAA
                AAAAGTACATATATGATCGTATATCC
SEQ ID NO: 640  GTATATACAAAAAAAAAAAGATATAAGTAAATATAAAAAAAA
                AAAATATATACATATATCGTATATAC
SEQ ID NO: 641  GTATATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAA
                AAAAGTACATATATGATCGTATATAC
SEQ ID NO: 642  GGATATACAAAAAAAAAAAGATAAATGAATATAAAAAAAAA
                AAATATATACATATATCGTATATCC
SEQ ID NO: 643  GTATATACAAAAAAAAAAAGATAAATGAATATAAAAAAAAA
                AAATATATACATATATCGTATATAC
SEQ ID NO: 644  GTATATACAAAAAAAAAAAGATGATATAAGTACAAAAAAAAA
                AAGTACATATATGATCGTATATAC
SEQ ID NO: 645  GTATATACAAAAAAAAAAATATAAATATATATTAAAAAAAAA
                AATATATATATATATAGTATATAC
SEQ ID NO: 646  GTATATACATAAAAAAAAAAAGATGATGTAAATATAAAAAAA
                AAAAATATATACATGATCATGTATATAC
SEQ ID NO: 647  GATATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAA
                AATATATATATATCGTATATC
SEQ ID NO: 648  GTGATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAA
                AATATATATATATCGTATCAC
SEQ ID NO: 649  GGTATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAA
                AATATATATATATCGTATACC
SEQ ID NO: 650  GGATATACATAAAAAAAAAAAGATAAATGAATAAAAAAAAA
                AAATATACATATATCATGTATATCC
SEQ ID NO: 651  GTATATACATAAAAAAAAAAAGATAAATGTATTAAAAAAAAA
                AATATACATATATCATGTATATAC
SEQ ID NO: 652  GTATATACATAAAAAAAAAAAGATGATAAATGTAAAAAAAAA
                AAACATATATGATCATGTATATAC
SEQ ID NO: 653  GATACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C
SEQ ID NO: 654  GACACTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*A*G*T*G*T*C
SEQ ID NO: 655  GATACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C
SEQ ID NO: 656  GGATCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*C
SEQ ID NO: 657  GACACTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 658  GGATCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*C
SEQ ID NO: 659  GCGTCTTTTTATTTTTTATATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*A*G*A*C*G*C
SEQ ID NO: 660  GCGTCTTTTTATTTTTGATATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*A*C*G*C
SEQ ID NO: 661  GTATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C
SEQ ID NO: 662  GTGATCTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*A*C
SEQ ID NO: 663  GTATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C
SEQ ID NO: 664  GTATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C
SEQ ID NO: 665  GGATACTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*C
SEQ ID NO: 666  GTGATCTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*A*C
SEQ ID NO: 667  GTGATCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*A*C
SEQ ID NO: 668  GGATACTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*C
SEQ ID NO: 669  GGATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*C
SEQ ID NO: 670  GCGATCTTTTTATTTTTTATATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*G*C
SEQ ID NO: 671  GCGATCTTTTTATTTTTGATATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*G*C
SEQ ID NO: 672  GCGATCTTTTTATTTTTGATATATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*G*C
SEQ ID NO: 673  GATATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C
SEQ ID NO: 674  GATATATTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*A*T*A*T*A*T*C
SEQ ID NO: 675  GATATATTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*A*T*A*T*A*T*C
SEQ ID NO: 676  GTGATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*A*C
SEQ ID NO: 677  GATATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 678  GATATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 679  GGTATACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C*C
SEQ ID NO: 680  GTGATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 681  GTGATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 682  GGTATACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 683  GGTATACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 684  GGTGTACTTTTTATTTTTTATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*G*T*A*C*A*C*C
SEQ ID NO: 685  GGTGTACTTTTTATTTTTGATATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 686  GGTGTACTTTTTATTTTTGATATATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 687  GTATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*A*C
SEQ ID NO: 688  GTATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 689  GTATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 690  GTATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 691  GTATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 692  GGATATACTTTTTATTTTTTATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C*C
SEQ ID NO: 693  GGATATACTTTTTATTTTTGATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 694  GGATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 695  GGATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 696  GGATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 697  GGTGATACTTTTTATTTTTTATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*G*T*A*T*C*A*C*C
SEQ ID NO: 698  GGTGATACTTTTTATTTTTGATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 699  GGTGATACTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 700  GGTGATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 701  GGTGATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 702  GGTGATCCTTTTTATTTTTTATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*G*G*A*T*C*A*C*C
SEQ ID NO: 703  GGTGATCCTTTTTATTTTTGATATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 704  GGTGATCCTTTTTATTTTTGATATATGTATATTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 705  GGTGATCCTTTTTATTTTTGATCATGTATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 706  GGTGATCCTTTTTATTTTTGATCATATATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 707  GATATATCACTTTTTATTTTTTATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*G*T*G*A*T*A*T*A*T*C
SEQ ID NO: 708  GTATATACATTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 709  GTATATACATTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 710  GTATATACATTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 711  GTATATACATTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 712  GGATATACACTTTTTATTTTTTATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 713  GGATATACATTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 714  GGATATACATTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 715  GGATATACATTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 716  GGATATACATTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 717  GGGTATATACTTTTTATTTTTTATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 718  GGATATACACTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 719  GGATATACACTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 720  GGATATACACTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 721  GGATATACACTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 722  GGGTATATACTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 723  GGGTATATACTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 724  GGGTATATACTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 725  GGGTATATACTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 726  GGATGTACACTTTTTATTTTTTATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 727  GGATGTACACTTTTTATTTTTGATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 728  GGATGTACACTTTTTATTTTTGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 729  GGATGTACACTTTTTATTTTTGATCATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 730  GGATGTACACTTTTTATTTTTGATCATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 731  GTATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATAC
SEQ ID NO: 732  GTATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 733  GGATATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATCC
SEQ ID NO: 734  GGATATACTTTTTATTTTTGATATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 735  GGTGATACTTTTTATTTTTTATATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*CACC
SEQ ID NO: 736  GGTGATACTTTTTATTTTTGATATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*CACC
SEQ ID NO: 737  GGTGATCCTTTTTATTTTTTATATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*G*A*T*CACC
SEQ ID NO: 738  GGTGATCCTTTTTATTTTTGATATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*G*A*T*CACC
SEQ ID NO: 739  GATATATCACTTTTTATTTTTTATATATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*A*TATATC
SEQ ID NO: 740  GTATATACATTTTTTATTTTTGATATATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 741  GGATATACACTTTTTATTTTTTATATATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*T*ATATCC
SEQ ID NO: 742  GGATATACATTTTTTATTTTTGATATATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 743  GGGTATATACTTTTTATTTTTTATATATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATACCC
SEQ ID NO: 744  GGATATACACTTTTTATTTTTGATATATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 745  GGGTATATACTTTTTATTTTTGATATATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 746  GTATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TAC
SEQ ID NO: 747  GTATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 748  GTATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 749  GGATATACTTTTTATTTTTTATATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TCC
SEQ ID NO: 750  GGATATACTTTTTATTTTTGATATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 751  GGATATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 752  GGTGATACTTTTTATTTTTTATATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*ACC
SEQ ID NO: 753  GGTGATACTTTTTATTTTTGATATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 754  GGTGATACTTTTTATTTTTGATATATGTATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 755  GGTGATCCTTTTTATTTTTTATATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*A*G*G*A*T*C*ACC
SEQ ID NO: 756  GGTGATCCTTTTTATTTTTGATATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 757  GGTGATCCTTTTTATTTTTGATATATGTATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 758  GATATATCACTTTTTATTTTTTATATATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*A*G*T*G*A*T*ATATC
SEQ ID NO: 759  GTATATACATTTTTTATTTTTGATATATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 760  GTATATACATTTTTTATTTTTGATATATGTATATATTTTTATTTT
                TTATA*T*A*C*A*T*A*T*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 761  GGATATACACTTTTTATTTTTTATATATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*A*G*T*G*T*A*TATCC
SEQ ID NO: 762  GGATATACATTTTTTATTTTTGATATATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 763  GGATATACATTTTTTATTTTTGATATATGTATATATTTTTATTTT
                TTATA*T*A*C*A*T*A*T*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 764  GGGTATATACTTTTTATTTTTTATATATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TACCC
SEQ ID NO: 765  GGATATACACTTTTTATTTTTGATATATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 766  GGATATACACTTTTTATTTTTGATATATGTATATATTTTTATTTT
                TTATA*T*A*C*A*T*A*T*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 767  GGGTATATACTTTTTATTTTTGATATATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 768  GGGTATATACTTTTTATTTTTGATATATGTATATATTTTTATTTT
                TTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 769  GATATATCACTTTTTATTTTTTATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*G*T*G*A*T*A*TATC
SEQ ID NO: 770  GTATATACATTTTTTATTTTTGATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATAC
SEQ ID NO: 771  GTATATACATTTTTTATTTTTGATATATGTATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*A*T*G*T*A*T*ATAC
SEQ ID NO: 772  GGATATACACTTTTTATTTTTTATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*G*T*G*T*A*T*ATCC
SEQ ID NO: 773  GGATATACATTTTTTATTTTTGATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 774  GGATATACATTTTTTATTTTTGATATATGTATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 775  GGGTATATACTTTTTATTTTTTATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*ACCC
SEQ ID NO: 776  GGATATACACTTTTTATTTTTGATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 777  GGATATACACTTTTTATTTTTGATATATGTATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 778  GGGTATATACTTTTTATTTTTGATATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 779  GGGTATATACTTTTTATTTTTGATATATGTATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 780  GATATATCACTTTTTATTTTTTATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*G*T*G*A*T*A*T*ATC
SEQ ID NO: 781  GTATATACATTTTTTATTTTTGATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 782  GTATATACATTTTTTATTTTTGATATATGTATATTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 783  GTATATACATTTTTTATTTTTGATCATGTATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 784  GTATATACATTTTTTATTTTTGATCATATATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 785  GGATATACACTTTTTATTTTTTATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*G*T*G*T*A*T*A*TCC
SEQ ID NO: 786  GGATATACATTTTTTATTTTTGATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 787  GGATATACATTTTTTATTTTTGATATATGTATATTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 788  GGATATACATTTTTTATTTTTGATCATGTATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 789  GGATATACATTTTTTATTTTTGATCATATATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 790  GGGTATATACTTTTTATTTTTTATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*G*T*A*T*A*T*A*CCC
SEQ ID NO: 791  GGATATACACTTTTTATTTTTGATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 792  GGATATACACTTTTTATTTTTGATATATGTATATTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 793  GGATATACACTTTTTATTTTTGATCATGTATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 794  GGATATACACTTTTTATTTTTGATCATATATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 795  GGGTATATACTTTTTATTTTTGATATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 796  GGGTATATACTTTTTATTTTTGATATATGTATATTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 797  GGGTATATACTTTTTATTTTTGATCATGTATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 798  GGGTATATACTTTTTATTTTTGATCATATATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 799  GTACATATATTTTTTTATTTTTGATATATATATTTTTATTTTTTA
                TA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 800  GTACATATATTTTTTTATTTTTGATATATGTATTTTTATTTTTTA
                CA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 801  GTACATATATTTTTTTATTTTTGATCATGTATTTTTTATTTTTAT
                AC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 802  GTACATATATTTTTTTATTTTTGATCATATATTTTTTATTTTTAT
                AT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 803  GATGTATATACTTTTTATTTTTTATATATATATTTTTATTTTTTA
                TA*T*A*T*A*T*A*G*T*A*T*A*T*A*C*A*TC
SEQ ID NO: 804  GGTACATATATTTTTTATTTTTGATATATATATTTTTATTTTTTA
                TA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 805  GGTACATATATTTTTTATTTTTGATATATGTATTTTTATTTTTTA
                CA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 806  GGTACATATATTTTTTATTTTTGATCATGTATTTTTTATTTTTAT
                AC*A*T*G*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 807  GGTACATATATTTTTTATTTTTGATCATATATTTTTTATTTTTAT
                AT*A*T*G*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 808  CGATCATATATTTTTTTATTTTTGATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 809  CGATCATATATTTTTTTATTTTTGATATATGTATTTTTATTTTTT
                ACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 810  CGATCATATATTTTTTTATTTTTGATCATGTATTTTTTATTTTTA
                TAC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 811  CGATCATATATTTTTTTATTTTTGATCATATATTTTTTATTTTTA
                TAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 812  GATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C
SEQ ID NO: 813  GACACTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*A*G*T*G*T*C
SEQ ID NO: 814  GATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C
SEQ ID NO: 815  GATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C
SEQ ID NO: 816  GATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT
                A*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C
SEQ ID NO: 817  GATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT
                A*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C
SEQ ID NO: 818  GGATCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*C
SEQ ID NO: 819  GACACTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 820  GACACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 821  GACACTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT
                A*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*C
SEQ ID NO: 822  GACACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT
                A*C*A*T*A*T*A*T*G*A*T*C*G*T*G*T*C
SEQ ID NO: 823  GGATCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*C
SEQ ID NO: 824  GGATCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*C
SEQ ID NO: 825  GGATCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT
                A*T*A*T*A*C*A*T*G*A*T*C*G*A*T*C*C
SEQ ID NO: 826  GGATCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT
                A*C*A*T*A*T*A*T*G*A*T*C*G*A*T*C*C
SEQ ID NO: 827  GCGTCTTTTTATTTTTTATAAATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*A*G*A*C*G*C
SEQ ID NO: 828  GCGTCTTTTTATTTTTGATAAATATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*A*C*G*C
SEQ ID NO: 829  GCGTCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*A*C*G*C
SEQ ID NO: 830  GCGTCTTTTTATTTTTGATGATGTATATATATTTTTATTTTTTAT
                A*T*A*T*A*C*A*T*G*A*T*C*G*A*C*G*C
SEQ ID NO: 831  GCGTCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTTAGT
                A*C*A*T*A*T*A*T*G*A*T*C*G*A*C*G*C
SEQ ID NO: 832  GTATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C
SEQ ID NO: 833  GTATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C
SEQ ID NO: 834  GTATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*C
SEQ ID NO: 835  GTATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTA
                C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*C
SEQ ID NO: 836  GTGATCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*A*C
SEQ ID NO: 837  GTGATCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*A*C
SEQ ID NO: 838  GTGATCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*G*A*T*C*G*A*T*C*A*C
SEQ ID NO: 839  GTGATCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTA
                C*A*T*A*T*A*T*G*A*T*C*G*A*T*C*A*C
SEQ ID NO: 840  GGATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*C
SEQ ID NO: 841  GGATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*C
SEQ ID NO: 842  GGATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*C
SEQ ID NO: 843  GGATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTA
                C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*C
SEQ ID NO: 844  GCGATCTTTTTATTTTTGATAAATATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*G*C
SEQ ID NO: 845  GCGATCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*G*C
SEQ ID NO: 846  GCGATCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*G*A*T*C*G*A*T*C*G*C
SEQ ID NO: 847  GCGATCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTGTA
                C*A*T*A*T*A*T*G*A*T*C*G*A*T*C*G*C
SEQ ID NO: 848  GATATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C
SEQ ID NO: 849  GATATATTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*A*T*A*T*A*T*C
SEQ ID NO: 850  GATATATTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*G*A*T*C*A*T*A*T*A*T*C
SEQ ID NO: 851  GATATATTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*A*T*A*T*A*T*C
SEQ ID NO: 852  GTGATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*A*C
SEQ ID NO: 853  GATATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 854  GATATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 855  GATATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 856  GGTATACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C*C
SEQ ID NO: 857  GTGATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 858  GTGATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 859  GTGATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 860  GTGATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 861  GGTATACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 862  GGTATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 863  GGTATACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 864  GGTATACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 865  GGTGTACTTTTTATTTTTTATAAATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*G*T*A*C*A*C*C
SEQ ID NO: 866  GGTGTACTTTTTATTTTTGATAAATATATATTTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 867  GGTGTACTTTTTATTTTTGATAAATGTATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 868  GGTGTACTTTTTATTTTTGATGATGTATATATTTTTATTTTTTAT
                A*T*A*C*A*T*G*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 869  GGTGTACTTTTTATTTTTGATGATATATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 870  GTATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 871  GTATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 872  GTATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 873  GTATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 874  GGATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 875  GGATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 876  GGATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 877  GGATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 878  GGTGATACTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 879  GGTGATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 880  GGTGATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 881  GGTGATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 882  GGTGATCCTTTTTATTTTTGATAAATATATATTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 883  GGTGATCCTTTTTATTTTTGATAAATGTATATTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 884  GGTGATCCTTTTTATTTTTGATGATGTATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 885  GGTGATCCTTTTTATTTTTGATGATATATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 886  GTATATACATTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 887  GTATATACATTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 888  GTATATACATTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 889  GTATATACATTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 890  GGATATACATTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 891  GGATATACATTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 892  GGATATACATTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 893  GGATATACATTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 894  GGATATACACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 895  GGATATACACTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 896  GGATATACACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 897  GGATATACACTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 898  GGGTATATACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 899  GGGTATATACTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 900  GGGTATATACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 901  GGGTATATACTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 902  GGATGTACACTTTTTATTTTTGATAAATATATTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 903  GGATGTACACTTTTTATTTTTGATAAATGTATTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 904  GGATGTACACTTTTTATTTTTGATGATGTATTTTTTATTTTTATA
                C*A*T*G*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 905  GGATGTACACTTTTTATTTTTGATGATATATTTTTTATTTTTATA
                T*A*T*G*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 906  GTATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATAC
SEQ ID NO: 907  GTATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 908  GTATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 909  GTATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTT
                TATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 910  GTATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT
                AGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 911  GGATATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATCC
SEQ ID NO: 912  GGATATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 913  GGATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 914  GGATATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTT
                TATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 915  GGATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT
                AGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 916  GGTGATACTTTTTATTTTTTATAAATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*CACC
SEQ ID NO: 917  GGTGATACTTTTTATTTTTGATAAATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*CACC
SEQ ID NO: 918  GGTGATACTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*CACC
SEQ ID NO: 919  GGTGATACTTTTTATTTTTGATGATGTATATATATTTTTATTTTT
                TATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*CACC
SEQ ID NO: 920  GGTGATACTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT
                AGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*CACC
SEQ ID NO: 921  GGTGATCCTTTTTATTTTTTATAAATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*G*A*T*CACC
SEQ ID NO: 922  GGTGATCCTTTTTATTTTTGATAAATATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*G*A*T*CACC
SEQ ID NO: 923  GGTGATCCTTTTTATTTTTGATAAATGTATATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*G*A*T*CACC
SEQ ID NO: 924  GGTGATCCTTTTTATTTTTGATGATGTATATATATTTTTATTTTT
                TATA*T*A*T*A*C*A*T*G*A*T*C*G*G*A*T*CACC
SEQ ID NO: 925  GGTGATCCTTTTTATTTTTGATGATATATGTACTTTTTTATTTTT
                AGTA*C*A*T*A*T*A*T*G*A*T*C*G*G*A*T*CACC
SEQ ID NO: 926  GATATATCACTTTTTATTTTTTATAAATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*A*TATATC
SEQ ID NO: 927  GTATATACATTTTTTATTTTTGATAAATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 928  GTATATACATTTTTTATTTTTGATAAATGTATATATTTTTTATTT
                TTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 929  GTATATACATTTTTTATTTTTGATGATGTATATATATTTTTATTT
                TTTATA*T*A*T*A*C*A*T*G*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 930  GTATATACATTTTTTATTTTTGATGATATATGTACTTTTTTATTT
                TTAGTA*C*A*T*A*T*A*T*G*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 931  GGATATACACTTTTTATTTTTTATAAATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*G*T*ATATCC
SEQ ID NO: 932  GGATATACATTTTTTATTTTTGATAAATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 933  GGATATACATTTTTTATTTTTGATAAATGTATATATTTTTTATTT
                TTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 934  GGATATACATTTTTTATTTTTGATGATGTATATATATTTTTATTT
                TTTATA*T*A*T*A*C*A*T*G*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 935  GGATATACATTTTTTATTTTTGATGATATATGTACTTTTTTATTT
                TTAGTA*C*A*T*A*T*A*T*G*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 936  GGGTATATACTTTTTATTTTTTATAAATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATACCC
SEQ ID NO: 937  GGATATACACTTTTTATTTTTGATAAATATATATATTTTTTATT
                TTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 938  GGATATACACTTTTTATTTTTGATAAATGTATATATTTTTTATT
                TTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 939  GGATATACACTTTTTATTTTTGATGATGTATATATATTTTTATT
                TTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 940  GGATATACACTTTTTATTTTTGATGATATATGTACTTTTTTATTT
                TTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 941  GGGTATATACTTTTTATTTTTGATAAATATATATATTTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 942  GGGTATATACTTTTTATTTTTGATAAATGTATATATTTTTTATTT
                TTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 943  GGGTATATACTTTTTATTTTTGATGATGTATATATATTTTTATTT
                TTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 944  GGGTATATACTTTTTATTTTTGATGATATATGTACTTTTTTATTT
                TTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 945  GTATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 946  GTATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 947  GTATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA
                TAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 948  GTATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTG
                TAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 949  GGATATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 950  GGATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 951  GGATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA
                TAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 952  GGATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTG
                TAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 953  GGTGATACTTTTTATTTTTGATAAATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 954  GGTGATACTTTTTATTTTTGATAAATGTATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 955  GGTGATACTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA
                TAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 956  GGTGATACTTTTTATTTTTGATGATATATGTACTTTTTATTTTTG
                TAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 957  GGTGATCCTTTTTATTTTTGATAAATATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 958  GGTGATCCTTTTTATTTTTGATAAATGTATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 959  GGTGATCCTTTTTATTTTTGATGATGTATATATTTTTTATTTTTA
                TAT*A*T*A*C*A*T*G*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 960  GGTGATCCTTTTTATTTTTGATGATATATGTACTTTTTATTTTTG
                TAC*A*T*A*T*A*T*G*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 961  GTATATACATTTTTTATTTTTGATAAATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 962  GTATATACATTTTTTATTTTTGATGATGTATATATTTTTTATTTT
                TATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 963  GTATATACATTTTTTATTTTTGATGATATATGTACTTTTTATTTT
                TGTAC*A*T*A*T*A*T*G*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 964  GGATATACATTTTTTATTTTTGATAAATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 965  GGATATACATTTTTTATTTTTGATAAATGTATATATTTTTATTTT
                TTATA*T*A*C*A*T*A*T*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 966  GGATATACATTTTTTATTTTTGATGATGTATATATTTTTTATTTT
                TATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 967  GGATATACATTTTTTATTTTTGATGATATATGTACTTTTTATTTT
                TGTAC*A*T*A*T*A*T*G*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 968  GGATATACACTTTTTATTTTTGATAAATATATATATTTTTATTT
                TTTATA*T*A*T*A*T*A*T*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 969  GGATATACACTTTTTATTTTTGATAAATGTATATATTTTTATTT
                TTTATA*T*A*C*A*T*A*T*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 970  GGATATACACTTTTTATTTTTGATGATGTATATATTTTTTATTTT
                TATAT*A*T*A*C*A*T*G*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 971  GGATATACACTTTTTATTTTTGATGATATATGTACTTTTTATTTT
                TGTAC*A*T*A*T*A*T*G*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 972  GGGTATATACTTTTTATTTTTGATAAATATATATATTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 973  GGGTATATACTTTTTATTTTTGATAAATGTATATATTTTTATTTT
                TTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 974  GGGTATATACTTTTTATTTTTGATGATGTATATATTTTTTATTTT
                TATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 975  GGGTATATACTTTTTATTTTTGATGATATATGTACTTTTTATTTT
                TGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 976  GATATATCACTTTTTATTTTTTATAAATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*G*T*G*A*T*A*TATC
SEQ ID NO: 977  GTATATACATTTTTTATTTTTGATAAATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATAC
SEQ ID NO: 978  GTATATACATTTTTTATTTTTGATGATGTATATATTTTTATTTTT
                TATA*T*A*C*A*T*G*A*T*C*A*T*G*T*A*T*ATAC
SEQ ID NO: 979  GTATATACATTTTTTATTTTTGATGATATATGTATTTTTATTTTT
                TACA*T*A*T*A*T*G*A*T*C*A*T*G*T*A*T*ATAC
SEQ ID NO: 980  GGATATACACTTTTTATTTTTTATAAATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*G*T*G*T*A*T*ATCC
SEQ ID NO: 981  GGATATACATTTTTTATTTTTGATAAATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 982  GGATATACATTTTTTATTTTTGATAAATGTATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 983  GGATATACATTTTTTATTTTTGATGATGTATATATTTTTATTTTT
                TATA*T*A*C*A*T*G*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 984  GGATATACATTTTTTATTTTTGATGATATATGTATTTTTATTTTT
                TACA*T*A*T*A*T*G*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 985  GGGTATATACTTTTTATTTTTTATAAATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*ACCC
SEQ ID NO: 986  GGATATACACTTTTTATTTTTGATAAATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 987  GGATATACACTTTTTATTTTTGATAAATGTATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 988  GGATATACACTTTTTATTTTTGATGATGTATATATTTTTATTTTT
                TATA*T*A*C*A*T*G*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 989  GGATATACACTTTTTATTTTTGATGATATATGTATTTTTATTTTT
                TACA*T*A*T*A*T*G*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 990  GGGTATATACTTTTTATTTTTGATAAATATATATTTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 991  GGGTATATACTTTTTATTTTTGATAAATGTATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 992  GGGTATATACTTTTTATTTTTGATGATGTATATATTTTTATTTTT
                TATA*T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 993  GGGTATATACTTTTTATTTTTGATGATATATGTATTTTTATTTTT
                TACA*T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 994  GTATATACATTTTTTATTTTTGATAAATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 995  GTATATACATTTTTTATTTTTGATGATGTATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 996  GTATATACATTTTTTATTTTTGATGATATATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 997  GGATATACATTTTTTATTTTTGATAAATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 998  GGATATACATTTTTTATTTTTGATAAATGTATATTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 999  GGATATACATTTTTTATTTTTGATGATGTATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1000 GGATATACATTTTTTATTTTTGATGATATATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1001 GGATATACACTTTTTATTTTTGATAAATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 1002 GGATATACACTTTTTATTTTTGATAAATGTATATTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 1003 GGATATACACTTTTTATTTTTGATGATGTATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 1004 GGATATACACTTTTTATTTTTGATGATATATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 1005 GGGTATATACTTTTTATTTTTGATAAATATATATTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 1006 GGGTATATACTTTTTATTTTTGATAAATGTATATTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 1007 GGGTATATACTTTTTATTTTTGATGATGTATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 1008 GGGTATATACTTTTTATTTTTGATGATATATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 1009 GTACATATATTTTTTTATTTTTGATAAATATATTTTTATTTTTTA
                TA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1010 GTACATATATTTTTTTATTTTTGATAAATGTATTTTTATTTTTTA
                CA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1011 GTACATATATTTTTTTATTTTTGATGATGTATTTTTTATTTTTAT
                AC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1012 GTACATATATTTTTTTATTTTTGATGATATATTTTTTATTTTTAT
                AT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1013 GGTACATATATTTTTTATTTTTGATAAATATATTTTTATTTTTTA
                TA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 1014 GGTACATATATTTTTTATTTTTGATAAATGTATTTTTATTTTTTA
                CA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 1015 GGTACATATATTTTTTATTTTTGATGATGTATTTTTTATTTTTAT
                AC*A*T*G*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 1016 GGTACATATATTTTTTATTTTTGATGATATATTTTTTATTTTTAT
                AT*A*T*G*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 1017 CGATCATATATTTTTTTATTTTTGATAAATATATTTTTATTTTTT
                ATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1018 CGATCATATATTTTTTTATTTTTGATAAATGTATTTTTATTTTTT
                ACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1019 CGATCATATATTTTTTTATTTTTGATGATGTATTTTTTATTTTTA
                TAC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1020 CGATCATATATTTTTTTATTTTTGATGATATATTTTTTATTTTTA
                TAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1021 GTATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT
                TATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1022 GTATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTT
                AGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1023 GGATATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT
                TATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1024 GGATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTT
                AGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1025 GGTGATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT
                TATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*CACC
SEQ ID NO: 1026 GGTGATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTT
                AGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*CACC
SEQ ID NO: 1027 GGTGATCCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTT
                TATA*T*A*T*A*C*A*T*G*A*T*C*G*G*A*T*CACC
SEQ ID NO: 1028 GGTGATCCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTT
                AGTA*C*A*T*A*T*A*T*G*A*T*C*G*G*A*T*CACC
SEQ ID NO: 1029 GTATATACATTTTTTATTTTTGATGATGTAAATATATTTTTATTT
                TTTATA*T*A*T*A*C*A*T*G*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 1030 GTATATACATTTTTTATTTTTGATGATATAAGTACTTTTTTATTT
                TTAGTA*C*A*T*A*T*A*T*G*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 1031 GGATATACATTTTTTATTTTTGATAAATGTAAATATTTTTTATT
                TTTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 1032 GGATATACATTTTTTATTTTTGATGATGTAAATATATTTTTATT
                TTTTATA*T*A*T*A*C*A*T*G*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 1033 GGATATACATTTTTTATTTTTGATGATATAAGTACTTTTTTATT
                TTTAGTA*C*A*T*A*T*A*T*G*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 1034 GGATATACACTTTTTATTTTTGATGATGTAAATATATTTTTATT
                TTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 1035 GGATATACACTTTTTATTTTTGATGATATAAGTACTTTTTTATT
                TTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 1036 GGGTATATACTTTTTATTTTTGATGATGTAAATATATTTTTATT
                TTTTATA*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 1037 GGGTATATACTTTTTATTTTTGATGATATAAGTACTTTTTTATT
                TTTAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 1038 GTATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA
                TAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1039 GTATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTG
                TAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1040 GGATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA
                TAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 1041 GGATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTT
                GTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 1042 GGTGATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA
                TAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 1043 GGTGATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTT
                GTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 1044 GGTGATCCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTA
                TAT*A*T*A*C*A*T*G*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 1045 GGTGATCCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTG
                TAC*A*T*A*T*A*T*G*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 1046 GTATATACATTTTTTATTTTTGATGATGTAAATATTTTTTATTTT
                TATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 1047 GTATATACATTTTTTATTTTTGATGATATAAGTACTTTTTATTTT
                TGTAC*A*T*A*T*A*T*G*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 1048 GGATATACATTTTTTATTTTTGATGATGTAAATATTTTTTATTTT
                TATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 1049 GGATATACATTTTTTATTTTTGATGATATAAGTACTTTTTATTT
                TTGTAC*A*T*A*T*A*T*G*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 1050 GGATATACACTTTTTATTTTTGATGATGTAAATATTTTTTATTT
                TTATAT*A*T*A*C*A*T*G*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 1051 GGATATACACTTTTTATTTTTGATGATATAAGTACTTTTTATTT
                TTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 1052 GGGTATATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTT
                TATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 1053 GGGTATATACTTTTTATTTTTGATGATATAAGTACTTTTTATTT
                TTGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 1054 GTATATACATTTTTTATTTTTGATGATATAAGTATTTTTATTTTT
                TACA*T*A*T*A*T*G*A*T*C*A*T*G*T*A*T*ATAC
SEQ ID NO: 1055 GGATATACATTTTTTATTTTTGATAAATGAATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 1056 GGATATACATTTTTTATTTTTGATGATATAAGTATTTTTATTTTT
                TACA*T*A*T*A*T*G*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 1057 GGATATACACTTTTTATTTTTGATAAATGAATATTTTTTATTTT
                TATAT*A*C*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 1058 GGATATACACTTTTTATTTTTGATGATATAAGTATTTTTATTTT
                TTACA*T*A*T*A*T*G*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 1059 GGGTATATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTT
                ATAT*A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 1060 GGGTATATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTT
                TACA*T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 1061 GTATATACATTTTTTATTTTTGATGATGAATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 1062 GGATATACATTTTTTATTTTTGATAAATGAATATTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1063 GGATATACATTTTTTATTTTTGATGATGAATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1064 GGATATACATTTTTTATTTTTGATGATAAATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1065 GGATATACACTTTTTATTTTTGATGATGAATATTTTTTATTTTT
                ATAT*A*C*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 1066 GGGTATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTA
                TAT*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 1067 GATACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTAT
                A*T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C
SEQ ID NO: 1068 GATACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT
                A*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C
SEQ ID NO: 1069 GACACTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTAT
                A*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*C
SEQ ID NO: 1070 GACACTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT
                A*C*A*T*A*T*A*T*G*A*T*C*G*T*G*T*C
SEQ ID NO: 1071 GGATCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTAT
                A*T*A*T*A*C*A*T*G*A*T*C*G*A*T*C*C
SEQ ID NO: 1072 GGATCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT
                A*C*A*T*A*T*A*T*G*A*T*C*G*A*T*C*C
SEQ ID NO: 1073 GCGTCTTTTTATTTTTGATGATGTAAATATATTTTTATTTTTTAT
                A*T*A*T*A*C*A*T*G*A*T*C*G*A*C*G*C
SEQ ID NO: 1074 GCGTCTTTTTATTTTTGATGATATAAGTACTTTTTTATTTTTAGT
                A*C*A*T*A*T*A*T*G*A*T*C*G*A*C*G*C
SEQ ID NO: 1075 GTATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*A*C
SEQ ID NO: 1076 GTATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA
                C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*C
SEQ ID NO: 1077 GTGATCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*G*A*T*C*G*A*T*C*A*C
SEQ ID NO: 1078 GTGATCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA
                C*A*T*A*T*A*T*G*A*T*C*G*A*T*C*A*C
SEQ ID NO: 1079 GGATACTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*C
SEQ ID NO: 1080 GGATACTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGT
                AC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*C
SEQ ID NO: 1081 GCGATCTTTTTATTTTTGATGATGTAAATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*G*A*T*C*G*A*T*C*G*C
SEQ ID NO: 1082 GCGATCTTTTTATTTTTGATGATATAAGTACTTTTTATTTTTGTA
                C*A*T*A*T*A*T*G*A*T*C*G*A*T*C*G*C
SEQ ID NO: 1083 GATATATTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*A*T*A*T*A*T*C
SEQ ID NO: 1084 GATATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 1085 GTGATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 1086 GTGATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 1087 GGTATACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 1088 GGTATACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 1089 GGTGTACTTTTTATTTTTGATAAATGAATATTTTTTATTTTTATA
                T*A*C*A*T*A*T*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 1090 GGTGTACTTTTTATTTTTGATGATATAAGTATTTTTATTTTTTAC
                A*T*A*T*A*T*G*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 1091 GTATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 1092 GTATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 1093 GGATATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 1094 GGTGATACTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 1095 GGTGATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTAC
                AT*A*T*A*T*G*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 1096 GGTGATCCTTTTTATTTTTGATGATGAATATTTTTTATTTTTATA
                T*A*C*A*T*G*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 1097 GATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C
SEQ ID NO: 1098 GATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C
SEQ ID NO: 1099 GACACTTTTTATTTTTGATATAAATATATAATTTTTATTTTTAT
                AT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 1100 GACACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTAT
                AT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 1101 GACACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTAT
                AT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 1102 GGATCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*C
SEQ ID NO: 1103 GGATCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*C
SEQ ID NO: 1104 GGATCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*C
SEQ ID NO: 1105 GCGTCTTTTTATTTTTGATATAAATATATAATTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*A*T*C*G*A*C*G*C
SEQ ID NO: 1106 GCGTCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*A*C*G*C
SEQ ID NO: 1107 GCGTCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTTATA
                T*A*T*A*C*A*T*A*T*A*T*C*G*A*C*G*C
SEQ ID NO: 1108 GTATATACATTTTTTATTTTTGATATAAATATATAATTTTTATTT
                TTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 1109 GTATATACATTTTTTATTTTTGATAAATGAATATATTTTTTATTT
                TTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 1110 GTATATACATTTTTTATTTTTGATATAAGTAAATATTTTTTATTT
                TTATAT*A*T*A*C*A*T*A*T*A*T*C*A*T*G*T*ATATAC
SEQ ID NO: 1111 GGATATACATTTTTTATTTTTGATATAAATATATAATTTTTATT
                TTTATAT*A*T*A*T*A*T*A*T*A*T*C*A*T*G*T*ATATCC
SEQ ID NO: 1112 GGATATACACTTTTTATTTTTGATATAAATATATAATTTTTATT
                TTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 1113 GGATATACACTTTTTATTTTTGATAAATGAATATATTTTTTATT
                TTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 1114 GGATATACACTTTTTATTTTTGATATAAGTAAATATTTTTTATT
                TTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*ATATCC
SEQ ID NO: 1115 GGGTATATACTTTTTATTTTTGATATAAATATATAATTTTTATT
                TTTATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 1116 GGGTATATACTTTTTATTTTTGATAAATGAATATATTTTTTATT
                TTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 1117 GGGTATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATT
                TTTATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATACCC
SEQ ID NO: 1118 GTATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C
SEQ ID NO: 1119 GTGATCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*A*C
SEQ ID NO: 1120 GTATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C
SEQ ID NO: 1121 GTATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C
SEQ ID NO: 1122 GTATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*C
SEQ ID NO: 1123 GGATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*C
SEQ ID NO: 1124 GTGATCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*A*C
SEQ ID NO: 1125 GTGATCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*A*C
SEQ ID NO: 1126 GTGATCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*A*C
SEQ ID NO: 1127 GGATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*C
SEQ ID NO: 1128 GGATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*C
SEQ ID NO: 1129 GGATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*C
SEQ ID NO: 1130 GCGATCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*G*C
SEQ ID NO: 1131 GCGATCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*G*C
SEQ ID NO: 1132 GCGATCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*A*T*C*G*A*T*C*G*C
SEQ ID NO: 1133 GCGATCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTTAT
                A*T*A*C*A*T*A*T*A*T*C*G*A*T*C*G*C
SEQ ID NO: 1134 GATATATCACTTTTTATTTTTTATAAATATATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*A*G*T*G*A*T*ATATC
SEQ ID NO: 1135 GTATATACATTTTTTATTTTTGATATAAATATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 1136 GTATATACATTTTTTATTTTTGATATATAAATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 1137 GGATATACACTTTTTATTTTTTATAAATATATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*A*G*T*G*T*A*TATCC
SEQ ID NO: 1138 GGATATACATTTTTTATTTTTGATATAAATATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 1139 GGATATACATTTTTTATTTTTGATATATAAATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 1140 GGATATACATTTTTTATTTTTGATAAATGAATATATTTTTATTT
                TTTATA*T*A*C*A*T*A*T*A*T*C*A*T*G*T*A*TATCC
SEQ ID NO: 1141 GGGTATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TACCC
SEQ ID NO: 1142 GGATATACACTTTTTATTTTTGATATAAATATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 1143 GGATATACACTTTTTATTTTTGATATATAAATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 1144 GGATATACACTTTTTATTTTTGATAAATGAATATATTTTTATTT
                TTTATA*T*A*C*A*T*A*T*A*T*C*G*T*G*T*A*TATCC
SEQ ID NO: 1145 GGGTATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 1146 GGGTATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTT
                TTATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 1147 GGGTATATACTTTTTATTTTTGATAAATGAATATATTTTTATTT
                TTTATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TACCC
SEQ ID NO: 1148 GATATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 1149 GTGATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 1150 GGTATACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 1151 GGTGTACTTTTTATTTTTGATAAATATATAATTTTTATTTTTATA
                T*A*T*A*T*A*T*A*T*C*G*T*A*C*A*C*C
SEQ ID NO: 1152 GTATATACATTTTTTATTTTTGATAAATATATAATTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATAC
SEQ ID NO: 1153 GGATATACATTTTTTATTTTTGATAAATATATAATTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*A*T*G*T*A*T*ATCC
SEQ ID NO: 1154 GGATATACACTTTTTATTTTTGATAAATATATAATTTTTATTTT
                TATAT*A*T*A*T*A*T*A*T*C*G*T*G*T*A*T*ATCC
SEQ ID NO: 1155 GGGTATATACTTTTTATTTTTGATAAATATATAATTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*ACCC
SEQ ID NO: 1156 GTATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 1157 GTATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*C
SEQ ID NO: 1158 GGATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 1159 GGATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 1160 GGATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTAC
                AT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*C*C
SEQ ID NO: 1161 GGTGATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 1162 GGTGATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*T*A*T*C*A*C*C
SEQ ID NO: 1163 GGTGATCCTTTTTATTTTTGATAAATATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 1164 GGTGATCCTTTTTATTTTTGATAAATGTATTTTTTTATTTTTTAT
                A*C*A*T*A*T*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 1165 GGTGATCCTTTTTATTTTTGATGATAAATGTTTTTTATTTTTACA
                T*A*T*A*T*G*A*T*C*G*G*A*T*C*A*C*C
SEQ ID NO: 1166 GTATATACATTTTTTATTTTTGATAAATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 1167 GTATATACATTTTTTATTTTTGATAAATGTATTTTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 1168 GTATATACATTTTTTATTTTTGATGATAAATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 1169 GGATATACATTTTTTATTTTTGATAAATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1170 GGATATACACTTTTTATTTTTGATAAATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 1171 GGATATACACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 1172 GGATATACACTTTTTATTTTTGATGATAAATGTTTTTTATTTTT
                ACAT*A*T*A*T*G*A*T*C*G*T*G*T*A*T*A*TCC
SEQ ID NO: 1173 GGGTATATACTTTTTATTTTTGATAAATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 1174 GGGTATATACTTTTTATTTTTGATAAATGTATTTTTTTATTTTTT
                ATA*C*A*T*A*T*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 1175 GGGTATATACTTTTTATTTTTGATGATAAATGTTTTTTATTTTTA
                CAT*A*T*A*T*G*A*T*C*G*T*A*T*A*T*A*CCC
SEQ ID NO: 1176 GTATATACATTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 1177 GTATATACATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 1178 GGATATACATTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1179 GGATATACATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                A*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1180 GGATATACATTTTTTATTTTTGATGATGAATTTTTTATTTTTATA
                C*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1181 GGATATACACTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1182 GGATATACACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1183 GGGTATATACTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 1184 GGGTATATACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 1185 GGATGTACACTTTTTATTTTTGATAAATATTTTTTTATTTTTTAT
                A*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 1186 GGATGTACACTTTTTATTTTTGATAAATGTTTTTTTATTTTTTAC
                A*T*A*T*A*T*C*G*T*G*T*A*C*A*T*C*C
SEQ ID NO: 1187 GTACATATATTTTTTTATTTTTGATAAATATTTTTTTATTTTTTA
                TA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1188 GTACATATATTTTTTTATTTTTGATAAATGTTTTTTTATTTTTTA
                CA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1189 GGTACATATATTTTTTATTTTTGATAAATATTTTTTTATTTTTTA
                TA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 1190 GGTACATATATTTTTTATTTTTGATAAATGTTTTTTTATTTTTTA
                CA*T*A*T*A*T*C*A*T*A*T*A*T*G*T*A*CC
SEQ ID NO: 1191 CGATCATATATTTTTTTATTTTTGATAAATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1192 CGATCATATATTTTTTTATTTTTGATAAATGTTTTTTTATTTTTT
                ACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1193 CGATCATATATTTTTTTATTTTTGATGATGAATTTTTTATTTTTA
                TAC*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1194 CGATCATATATTTTTTTATTTTTGATGATAAATTTTTTATTTTTA
                TAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1195 GTATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1196 GTATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1197 GGATATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1198 GGATATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1199 GGTGATACTTTTTATTTTTGATATAAATATATAATTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*CACC
SEQ ID NO: 1200 GGTGATACTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*CACC
SEQ ID NO: 1201 GGTGATCCTTTTTATTTTTGATATAAATATATAATTTTTATTTTT
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*G*A*T*CACC
SEQ ID NO: 1202 GGTGATCCTTTTTATTTTTGATAAATGAATATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*G*A*T*CACC
SEQ ID NO: 1203 GTATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1204 GGATATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1205 GGTGATACTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*CACC
SEQ ID NO: 1206 GGTGATCCTTTTTATTTTTGATATAAGTAAATATTTTTTATTTTT
                ATAT*A*T*A*C*A*T*A*T*A*T*C*G*G*A*T*CACC
SEQ ID NO: 1207 GTATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TAC
SEQ ID NO: 1208 GTATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1209 GTATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1210 GGATATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TCC
SEQ ID NO: 1211 GGATATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 1212 GGATATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 1213 GGTGATACTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C*ACC
SEQ ID NO: 1214 GGTGATACTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 1215 GGTGATACTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 1216 GGTGATCCTTTTTATTTTTTATAAATATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*A*G*G*A*T*C*ACC
SEQ ID NO: 1217 GGTGATCCTTTTTATTTTTGATATAAATATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 1218 GGTGATCCTTTTTATTTTTGATAAATGAATATATTTTTATTTTTT
                ATA*T*A*C*A*T*A*T*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 1219 GTATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1220 GGATATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 1221 GGTGATACTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 1222 GGTGATCCTTTTTATTTTTGATATATAAATATTTTTTTATTTTTT
                ATA*T*A*T*A*T*A*T*A*T*C*G*G*A*T*C*ACC
SEQ ID NO: 1223 GATACAAAAAAAAAAATATATATATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C
SEQ ID NO: 1224 GACACAAAAAAAAAAAGATATATATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 1225 GATATACAAAAAAAAAAATATATATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C
SEQ ID NO: 1226 GATATATAAAAAAAAAAAGATATATGTATATAAAAAAAAAAA
                ATAT*A*C*A*T*A*T*A*T*C*A*T*A*T*A*T*C
SEQ ID NO: 1227 GATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 1228 GGTATACAAAAAAAAAAATATATATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*C*C
SEQ ID NO: 1229 GATATATCACAAAAAAAAAAATATATATATAAAAAAAAAAAA
                TATA*T*A*T*A*T*A*G*T*G*A*T*A*T*A*T*C
SEQ ID NO: 1230 GTATATACATAAAAAAAAAAAGATATATGTAAAAAAAAAAAA
                TACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 1231 GGATATACATAAAAAAAAAAAGATATATGTAAAAAAAAAAA
                ATACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1232 GGATATACATAAAAAAAAAAAGATCATGTATAAAAAAAAAAA
                ATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1233 GGGTATATACAAAAAAAAAAATATATATATAAAAAAAAAAAA
                TATA*T*A*T*A*T*A*G*T*A*T*A*T*A*C*C*C
SEQ ID NO: 1234 GTATATACAAAAAAAAAAATATATATATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATAC
SEQ ID NO: 1235 GTATATACAAAAAAAAAAAGATATATATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1236 GGATATACAAAAAAAAAAATATATATATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATCC
SEQ ID NO: 1237 GGATATACAAAAAAAAAAAGATATATATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1238 GTATATACAAAAAAAAAAATATATATATATATAAAAAAAAAA
                AATATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TAC
SEQ ID NO: 1239 GTATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAA
                AATATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1240 GGATATACAAAAAAAAAAATATATATATATATAAAAAAAAAA
                AATATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TCC
SEQ ID NO: 1241 GGATATACAAAAAAAAAAAGATATATATATATAAAAAAAAAA
                AATATA*T*A*T*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 1242 GATATATCACAAAAAAAAAAATATATATATATAAAAAAAAAA
                AATATA*T*A*T*A*T*A*T*A*G*T*G*A*T*A*T*ATC
SEQ ID NO: 1243 GGATATACATAAAAAAAAAAAGATATATATATAAAAAAAAAA
                AATATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1244 GTACATATATTAAAAAAAAAAAGATATATATAAAAAAAAAAA
                ATATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1245 GATGTATATACAAAAAAAAAAATATATATATAAAAAAAAAAA
                ATATA*T*A*T*A*T*A*G*T*A*T*A*T*A*C*A*TC
SEQ ID NO: 1246 CGATCATATATTAAAAAAAAAAAGATATATATAAAAAAAAAA
                AATATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1247 CGATCATATATTAAAAAAAAAAAGATATATGTAAAAAAAAAA
                AATACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*A*TCG
SEQ ID NO: 1248 GATACAAAAAAAAAAATATAAATATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*C
SEQ ID NO: 1249 GGATCAAAAAAAAAAATATAAATATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*A*T*A*G*A*T*C*C
SEQ ID NO: 1250 GACACAAAAAAAAAAAGATAAATATATATATAAAAAAAAAA
                AATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 1251 GACACAAAAAAAAAAAGATGATGTATATATAAAAAAAAAAA
                ATATA*T*A*T*A*C*A*T*G*A*T*C*G*T*G*T*C
SEQ ID NO: 1252 GCGTCAAAAAAAAAAAGATAAATATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*A*T*C*G*A*C*G*C
SEQ ID NO: 1253 GATATACAAAAAAAAAAATATAAATATATATAAAAAAAAAAA
                ATAT*A*T*A*T*A*T*A*T*A*G*T*A*T*A*T*C
SEQ ID NO: 1254 GTATATACATAAAAAAAAAAAGATAAATGTAAAAAAAAAAA
                ATACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 1255 GTATATACATAAAAAAAAAAAGATGATATATAAAAAAAAAAA
                ATAT*A*T*G*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 1256 GGATATACATAAAAAAAAAAAGATAAATATAAAAAAAAAAA
                ATATA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1257 GGATATACATAAAAAAAAAAAGATGATATATAAAAAAAAAA
                AATAT*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1258 GTATATACAAAAAAAAAAATATAAATATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATAC
SEQ ID NO: 1259 GTATATACAAAAAAAAAAAGATAAATATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1260 GGATATACAAAAAAAAAAATATAAATATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*T*A*G*T*A*T*ATCC
SEQ ID NO: 1261 GGATATACAAAAAAAAAAAGATAAATATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1262 GTATATACAAAAAAAAAAAGATAAATGTATATAAAAAAAAAA
                AATATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1263 GGATATACAAAAAAAAAAAGATAAATGTATATAAAAAAAAA
                AAATATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 1264 GGTGATACAAAAAAAAAAAGATGATGTATATATAAAAAAAAA
                AAATAT*A*T*A*C*A*T*G*A*T*C*G*T*A*T*C*ACC
SEQ ID NO: 1265 GATATATCACAAAAAAAAAAATATAAATATATATAAAAAAAA
                AAAATAT*A*T*A*T*A*T*A*T*A*G*T*G*A*T*A*TATC
SEQ ID NO: 1266 GTATATACATAAAAAAAAAAAGATAAATATATAAAAAAAAAA
                AATATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 1267 GTATATACATAAAAAAAAAAAGATGATATATGTAAAAAAAAA
                AAACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 1268 GGATATACATAAAAAAAAAAAGATAAATATATAAAAAAAAA
                AAATATA*T*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1269 GTACATATATTAAAAAAAAAAAGATAAATATAAAAAAAAAAA
                ATATA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1270 GTACATATATTAAAAAAAAAAAGATAAATGTAAAAAAAAAAA
                ATACA*T*A*T*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1271 GTACATATATTAAAAAAAAAAAGATGATATATAAAAAAAAAA
                AATAT*A*T*G*A*T*C*A*A*T*A*T*A*T*G*T*AC
SEQ ID NO: 1272 GGATATACATAAAAAAAAAAAGATGATGAATAAAAAAAAAA
                AATAC*A*T*G*A*T*C*A*T*G*T*A*T*A*T*C*C
SEQ ID NO: 1273 GTATATACATAAAAAAAAAAAGATAAATGTTAAAAAAAAAAA
                TACA*T*A*T*A*T*C*A*T*G*T*A*T*A*T*A*C
SEQ ID NO: 1274 GATACAAAAAAAAAAAGATATAAATATATAAAAAAAAAAAA
                AATAT*A*T*A*T*A*T*A*T*A*T*C*G*T*A*T*C
SEQ ID NO: 1275 GATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAAAA
                AAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*C
SEQ ID NO: 1276 GACACAAAAAAAAAAAGATAAATGAATATATAAAAAAAAAA
                AATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*G*T*C
SEQ ID NO: 1277 GGATATACAAAAAAAAAAAGATATAAGTAAATATAAAAAAA
                AAAAATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1278 GGATATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAA
                AAAAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATCC
SEQ ID NO: 1279 GTATATACAAAAAAAAAAAGATATAAGTAAATATAAAAAAAA
                AAAATAT*A*T*A*C*A*T*A*T*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1280 GTATATACAAAAAAAAAAAGATGATATAAGTACTAAAAAAAA
                AAAAGTA*C*A*T*A*T*A*T*G*A*T*C*G*T*A*T*ATAC
SEQ ID NO: 1281 GGATATACAAAAAAAAAAAGATAAATGAATATAAAAAAAAA
                AAATATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TCC
SEQ ID NO: 1282 GTATATACAAAAAAAAAAAGATAAATGAATATAAAAAAAAA
                AAATATA*T*A*C*A*T*A*T*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1283 GTATATACAAAAAAAAAAAGATGATATAAGTACAAAAAAAAA
                AAGTAC*A*T*A*T*A*T*G*A*T*C*G*T*A*T*A*TAC
SEQ ID NO: 1284 GTATATACAAAAAAAAAAATATAAATATATATTAAAAAAAAA
                AATATA*T*A*T*A*T*A*T*A*T*A*G*T*A*T*A*TAC
SEQ ID NO: 1285 GTATATACATAAAAAAAAAAAGATGATGTAAATATAAAAAAA
                AAAAATAT*A*T*A*C*A*T*G*A*T*C*A*T*G*T*A*TATAC
SEQ ID NO: 1286 GATATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAA
                AATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*T*C
SEQ ID NO: 1287 GTGATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAA
                AATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*C*A*C
SEQ ID NO: 1288 GGTATACAAAAAAAAAAAGATAAATATATAAAAAAAAAAAA
                AATAT*A*T*A*T*A*T*A*T*C*G*T*A*T*A*C*C
SEQ ID NO: 1289 GGATATACATAAAAAAAAAAAGATAAATGAATAAAAAAAAA
                AAATATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TCC
SEQ ID NO: 1290 GTATATACATAAAAAAAAAAAGATAAATGTATTAAAAAAAAA
                AATATA*C*A*T*A*T*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 1291 GTATATACATAAAAAAAAAAAGATGATAAATGTAAAAAAAAA
                AAACAT*A*T*A*T*G*A*T*C*A*T*G*T*A*T*A*TAC
SEQ ID NO: 1292 TATATATTATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC
                AATATATA
SEQ ID NO: 1293 GATATATTATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC
                AATATATC
SEQ ID NO: 1294 GATATATCATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC
                GATATATC
SEQ ID NO: 1295 GATATGTCATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC
                GACATATC
SEQ ID NO: 1296 GTGATGTCATTTTATTTTAATCGAGTCTTTTTGACTCGATATAC
                GACATCAC
SEQ ID NO: 1297 TATATATTATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC
                AATATATA
SEQ ID NO: 1298 GATATATTATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC
                AATATATC
SEQ ID NO: 1299 GATATATCATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC
                GATATATC
SEQ ID NO: 1300 GATATGTCATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC
                GACATATC
SEQ ID NO: 1301 GTGATGTCATTTTATTTTATGCGAGTCTTTTTGACTCGCAGCCC
                GACATCAC
SEQ ID NO: 1302 TATATATTATTTTATTTTAGTATATCGGACTCGATATACAATAT
                ATA
SEQ ID NO: 1303 GATATATTATTTTATTTTAGTATATCGGACTCGATATACAATAT
                ATC
SEQ ID NO: 1304 GATATATCATTTTATTTTAGTATATCGGACTCGATATACGATAT
                ATC
SEQ ID NO: 1305 GATATGTCATTTTATTTTAGTATATCGGACTCGATATACGACAT
                ATC
SEQ ID NO: 1306 GTGATGTCATTTTATTTTAGTATATCGGACTCGATATACGACAT
                CAC
SEQ ID NO: 1307 TATATATTATTTTATTTTAGGGCTGCGGACTCGCAGCCCAATAT
                ATA
SEQ ID NO: 1308 GATATATTATTTTATTTTAGGGCTGCGGACTCGCAGCCCAATA
                TATC
SEQ ID NO: 1309 GATATATCATTTTATTTTAGGGCTGCGGACTCGCAGCCCGATA
                TATC
SEQ ID NO: 1310 GATATGTCATTTTATTTTAGGGCTGCGGACTCGCAGCCCGACA
                TATC
SEQ ID NO: 1311 GTGATGTCATTTTATTTTAGGGCTGCGGACTCGCAGCCCGACA
                TCAC
SEQ ID NO: 1312 TATATATATTATTACTATATGGACTCGCATATAGATATATA
SEQ ID NO: 1313 GATATATATTATTACTATATGGACTCGCATATAGATATATC
SEQ ID NO: 1314 GATATACATTATTACTATATGGACTCGCATATAGGTATATC
SEQ ID NO: 1315 GATATCCATTATTACTATATGGACTCGCATATAGGGATATC
SEQ ID NO: 1316 GTGATACATTATTACTATATGGACTCGCATATAGGTATCAC
SEQ ID NO: 1317 TATATATTTTATTTCGGGCTGGACTCGCAGCCCGATATATA
SEQ ID NO: 1318 GATATATATTATTACGGGCTGGACTCGCAGCCCGATATATC
SEQ ID NO: 1319 GATATACATTATTACGGGCTGGACTCGCAGCCCGGTATATC
SEQ ID NO: 1320 GATATCCATTATTACGGGCTGGACTCGCAGCCCGGGATATC
SEQ ID NO: 1321 GTGATACATTATTACGGGCTGGACTCGCAGCCCGGTATCAC
SEQ ID NO: 1322 TATATATTTTATTTCTATATGTTTATTTCGAGTCTTTTGACTCGC
                ATATAGATATATA
SEQ ID NO: 1323 GATATATATTATTACTATATGATTATTACGAGTCTTTTGACTCG
                CATATAGATATATC
SEQ ID NO: 1324 GATATACATTATTACTATATGATTATTACGAGTCTTTTGACTCG
                CATATAGGTATATC
SEQ ID NO: 1325 GATATCCATTATTACTATATGATTATTACGAGTCTTTTGACTCG
                CATATAGGGATATC
SEQ ID NO: 1326 GTGATACATTATTACTATATGATTATTACGAGTCTTTTGACTCG
                CATATAGGTATCAC
SEQ ID NO: 1327 TATATATATTATTACGGGCTGATTATTACGAGTCTTTTGACTCG
                CAGCCCGATATATA
SEQ ID NO: 1328 GATATATATTATTACGGGCTGATTATTACGAGTCTTTTGACTCG
                CAGCCCGATATATC
SEQ ID NO: 1329 GATATACATTATTACGGGCTGATTATTACGAGTCTTTTGACTC
                GCAGCCCGGTATATC
SEQ ID NO: 1330 GATATCCATTATTACGGGCTGATTATTACGAGTCTTTTGACTCG
                CAGCCCGGGATATC
SEQ ID NO: 1331 GTGATACATTATTACGGGCTGATTATTACGAGTCTTTTGACTC
                GCAGCCCGGTATCAC
SEQ ID NO: 1332 TATATATTTATTTCATATCGACTCGCAGATATGTATATA
SEQ ID NO: 1333 GATATCATTATTACATATCGACTCGCAGATATGGATATC
SEQ ID NO: 1334 GTGATCATTATTACATATCGACTCGCAGATATGGATCAC
SEQ ID NO: 1335 GTGTGCATTATTACATATCGACTCGCAGATATGGCACAC
SEQ ID NO: 1336 GATATCATTATTACCGGGCGACTCGCAGCCCGGGATATC
SEQ ID NO: 1337 GTGATCATTATTACCGGGCGACTCGCAGCCCGGGATCAC
SEQ ID NO: 1338 GTGTGCATTATTACCGGGCGACTCGCAGCCCGGGCACAC
SEQ ID NO: 1339 TATATATTTATTTCATATCTTTATTTTGCGAGTCTTTTGACTCGC
                AGATATGTATATA
SEQ ID NO: 1340 GATATCATTATTACATATCATTATTATGCGAGTCTTTTGACTCG
                CAGATATGGATATC
SEQ ID NO: 1341 GTGATCATTATTACATATCATTATTATGCGAGTCTTTTGACTCG
                CAGATATGGATCAC
SEQ ID NO: 1342 GTGTGCATTATTACATATCATTATTATGCGAGTCTTTTGACTCG
                CAGATATGGCACAC
SEQ ID NO: 1343 GATATCATTATTACCGGGCATTATTATGCGAGTCTTTTGACTCG
                CAGCCCGGGATATC
SEQ ID NO: 1344 GTGATCATTATTACCGGGCATTATTATGCGAGTCTTTTGACTCG
                CAGCCCGGGATCAC
SEQ ID NO: 1345 GTGTGCATTATTACCGGGCATTATTATGCGAGTCTTTTGACTCG
                CAGCCCGGGCACAC
SEQ ID NO: 1346 GTATGATTATTACACAGGACTCGCAGCCTGTGCATAC
SEQ ID NO: 1347 GTGTGATTATTACACAGGACTCGCAGCCTGTGCACAC
SEQ ID NO: 1348 GTATGATTATTACCCGGGACTCGCAGCCCGGGCATAC
SEQ ID NO: 1349 GTGTGATTATTACCCGGGACTCGCAGCCCGGGCACAC
SEQ ID NO: 1350 GTATGATTATTACACAGATTATTAGCTGCATTATTAGAGTCTTT
                TGACTCGCAGCCTGTGCATAC
SEQ ID NO: 1351 GTGTGATTATTACACAGATTATTAGCTGCATTATTAGAGTCTTT
                TGACTCGCAGCCTGTGCACAC
SEQ ID NO: 1352 TATATATTATTACCCGGATTATTAGCTGCATTATTAGAGTCTTT
                TGACTCGCAGCCCGGGATATA
SEQ ID NO: 1353 GATATATTATTACCCGGATTATTAGCTGCATTATTAGAGTCTTT
                TGACTCGCAGCCCGGGATATC
SEQ ID NO: 1354 GTATGATTATTACCCGGATTATTAGCTGCATTATTAGAGTCTTT
                TGACTCGCAGCCCGGGCATAC
SEQ ID NO: 1355 GTGTGATTATTACCCGGATTATTAGCTGCATTATTAGAGTCTTT
                TGACTCGCAGCCCGGGCACAC
SEQ ID NO: 1356 GACTCGATATACAATATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATAATTATTATTGTATAT
SEQ ID NO: 1357 GACTCGATATACAATATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATAATTATTATTGTATAT
SEQ ID NO: 1358 GACTCGATATACGATATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATAATTATTATCGTATAT
SEQ ID NO: 1359 GACTCGATATACGACATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATGATTATTATCGTATAT
SEQ ID NO: 1360 GACTCGATATACGACATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATGATTATTATCGTATAT
SEQ ID NO: 1361 GACTCGCAGCCCAATATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATAATTATTATTGGGCTG
SEQ ID NO: 1362 GACTCGCAGCCCAATATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATAATTATTATTGGGCTG
SEQ ID NO: 1363 GACTCGCAGCCCGATATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATAATTATTATCGGGCTG
SEQ ID NO: 1364 GACTCGCAGCCCGACATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATGATTATTATCGGGCTG
SEQ ID NO: 1365 GACTCGCAGCCCGACATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATGATTATTATCGGGCTG
SEQ ID NO: 1366 GACTCGATATACAATATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATAATTATTATTGTATATTATTAATCGAGTC
SEQ ID NO: 1367 GACTCGATATACAATATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATAATTATTATTGTATATTATTAATCGAGTC
SEQ ID NO: 1368 GACTCGATATACGATATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATAATTATTATCGTATATTATTAATCGAGTC
SEQ ID NO: 1369 GACTCGATATACGACATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATGATTATTATCGTATATTATTAATCGAGTC
SEQ ID NO: 1370 GACTCGATATACGACATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATGATTATTATCGTATATTATTAATCGAGTC
SEQ ID NO: 1371 GACTCGCAGCCCAATATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATAATTATTATTGGGCATTATTATGCGAGTC
SEQ ID NO: 1372 GACTCGCAGCCCAATATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATAATTATTATTGGGCATTATTATGCGAGTC
SEQ ID NO: 1373 GACTCGCAGCCCGATATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATAATTATTATCGGGCATTATTATGCGAGTC
SEQ ID NO: 1374 GACTCGCAGCCCGACATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATGATTATTATCGGGCATTATTATGCGAGTC
SEQ ID NO: 1375 GACTCGCAGCCCGACATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATGATTATTATCGGGCATTATTATGCGAGTC
SEQ ID NO: 1376 GACTCGCATATAGATATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATATTATTAATCTATA
SEQ ID NO: 1377 GACTCGCATATAGATATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATATTATTAATCTATA
SEQ ID NO: 1378 GACTCGCATATAGGTATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATATTATTAACCTATA
SEQ ID NO: 1379 GACTCGCATATAGGGATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATATTATTACCCTATA
SEQ ID NO: 1380 GACTCGCATATAGGTATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATATTATTAACCTATA
SEQ ID NO: 1381 GACTCGCAGCCCGATATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATATTATTAATCGGGC
SEQ ID NO: 1382 GACTCGCAGCCCGATATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATATTATTAATCGGGC
SEQ ID NO: 1383 GACTCGCAGCCCGGTATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATATTATTAACCGGGC
SEQ ID NO: 1384 GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATATTATTACCCGGGC
SEQ ID NO: 1385 GACTCGCAGCCCGGTATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATATTATTAACCGGGC
SEQ ID NO: 1386 GACTCGCATATAGATATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATATTATTAATCTATAATTATTATGCGAGT
SEQ ID NO: 1387 GACTCGCATATAGATATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATATTATTAATCTATAATTATTATGCGAGT
SEQ ID NO: 1388 GACTCGCATATAGGTATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATATTATTAACCTATAATTATTATGCGAGT
SEQ ID NO: 1389 GACTCGCATATAGGGATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATATTATTACCCTATAATTATTATGCGAGT
SEQ ID NO: 1390 GACTCGCATATAGGTATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATATTATTAACCTATAATTATTATGCGAGT
SEQ ID NO: 1391 GACTCGCAGCCCGATATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATATTATTAATCGGGCATTATTATGCGAGT
SEQ ID NO: 1392 GACTCGCAGCCCGATATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATATTATTAATCGGGCATTATTATGCGAGT
SEQ ID NO: 1393 GACTCGCAGCCCGGTATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATATTATTAACCGGGCATTATTATGCGAGT
SEQ ID NO: 1394 GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATATTATTACCCGGGCATTATTATGCGAGT
SEQ ID NO: 1395 GACTCGCAGCCCGGTATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATATTATTAACCGGGCATTATTATGCGAGT
SEQ ID NO: 1396 GACTCGCAGATATGTATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATAATTATTATACATA
SEQ ID NO: 1397 GACTCGCAGATATGTATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATAATTATTATACATA
SEQ ID NO: 1398 GACTCGCAGATATGGATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATAATTATTATCCATA
SEQ ID NO: 1399 GACTCGCAGATATGGATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGAATTATTATCCATA
SEQ ID NO: 1400 GACTCGCAGATATGGCACACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGTATTATTAGCCATA
SEQ ID NO: 1401 GACTCGCAGCCCGGTATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATAATTATTATACCGG
SEQ ID NO: 1402 GACTCGCAGCCCGGTATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATAATTATTATACCGG
SEQ ID NO: 1403 GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATAATTATTATCCCGG
SEQ ID NO: 1404 GACTCGCAGCCCGGGATCACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGAATTATTATCCCGG
SEQ ID NO: 1405 GACTCGCAGCCCGGGCACACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGTATTATTAGCCCGG
SEQ ID NO: 1406 GACTCGCAGCCTGTGATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATTATTAATCAC
SEQ ID NO: 1407 GACTCGCAGCCTGTGATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATTATTAATCAC
SEQ ID NO: 1408 GACTCGCAGCCTGTGCATACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTAATTATTATGCAC
SEQ ID NO: 1409 GACTCGCAGCCTGTGCACACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATTATTATGCAC
SEQ ID NO: 1410 GACTCGCAGCCCGGGATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATTATTAATCCC
SEQ ID NO: 1411 GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATTATTAATCCC
SEQ ID NO: 1412 GACTCGCAGCCCGGGCATACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTAATTATTATGCCC
SEQ ID NO: 1413 GACTCGCAGCCCGGGCACACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATTATTATGCCC
SEQ ID NO: 1414 GACTCGCAGCCTGTGATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATTATTAATCACATTATTAAGGCTATTATTAGCG
                AG
SEQ ID NO: 1415 GACTCGCAGCCTGTGATATCGCGCGCGCAATAAGCGCGCATTA
                TTAGCGATATTATTAATCACATTATTAAGGCTATTATTAGCGA
                G
SEQ ID NO: 1416 GACTCGCAGCCTGTGCATACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTAATTATTATGCACATTATTAAGGCTATTATTAGCG
                AG
SEQ ID NO: 1417 GACTCGCAGCCTGTGCACACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATTATTATGCACATTATTAAGGCTATTATTAGCG
                AG
SEQ ID NO: 1418 GACTCGCAGCCCGGGATATAGCGCGCGCAATAAGCGCGCATT
                ATTAGCTATATTATTAATCCCATTATTAGGGCTATTATTAGCGA
                G
SEQ ID NO: 1419 GACTCGCAGCCCGGGATATCGCGCGCGCAATAAGCGCGCATT
                ATTAGCGATATTATTAATCCCATTATTAGGGCTATTATTAGCG
                AG
SEQ ID NO: 1420 GACTCGCAGCCCGGGCATACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTAATTATTATGCCCATTATTAGGGCTATTATTAGCG
                AG
SEQ ID NO: 1421 GACTCGCAGCCCGGGCACACGCGCGCGCAATAAGCGCGCATT
                ATTAGCGTGATTATTATGCCCATTATTAGGGCTATTATTAGCG
                AG
*indicate the bonds that are phosphorothioate (PS) modified. These sequences may include nuclease resistant modifications such as PS modifications in all bases except the Loop sequences, where Loop sequences are the unhybridized bases. The number of modifications, e.g., PS, can vary from “0” to “max = total number of bases-number of bases in loops.

In any of the foregoing embodiments, the blocked nucleic acid molecules of the disclosure may further contain a reporter moiety attached thereto such that cleavage of the blocked nucleic acid releases a signal from the reporter moiety. (See FIG. 4, mechanisms depicted at center and bottom.)

Also, in any of the foregoing embodiments, the blocked nucleic acid molecule may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the blocked nucleic acid molecules of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blocked nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-0-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, any other nucleic acid molecule modifications described above, and any combination thereof.

FIG. 2G at left shows an exemplary single-strand blocked nucleic acid molecule and how the configuration of this blocked nucleic acid molecule is able to block

R-loop formation with an RNP complex, thereby blocking activation of the trans-cleavage activity of RNP2. The single-strand blocked nucleic acid molecule is self-hybridized and comprises: a target strand (TS) sequence complementary to the gRNA (e.g., crRNA) of RNP2; a cleavable non-target strand (NTS) sequence that is partially hybridized (e.g., it contains secondary loop structures) to the TS sequence; and a protospacer adjacent motif (PAM) sequence (e.g., 5′ NAAA 3′) that is specifically located at the 3′ end of the TS sequence. An RNP complex with 3′→5′ diffusion (e.g., 1D diffusion) initiates R-loop formation upon PAM recognition. R-loop formation is completed upon a stabilizing ≤17 base hybridization of the TS to the gRNA of RNP2; however, because of the orientation of the PAM sequence relative to the secondary loop structure(s), the blocked nucleic acid molecule sterically prevents the TS sequence from hybridizing with the gRNA of RNP2, thereby blocking the stable R-loop formation required for the cascade reaction.

FIG. 2G at right shows the blocked nucleic acid molecule being unblocked via trans-cleavage (e.g., by RNP1) and subsequent dehybridization of the NTS's secondary loop structures, followed by binding of the TS sequence to the gRNA of RNP2, thereby completing stable R-loop formation and activating the trans-cleavage activity of the RNP2 complex.

In some embodiments, the blocked nucleic acid molecules provided herein are circular DNAs, RNAs or chimeric (DNA-RNA) molecules (FIG. 2H), and the blocked nucleic acid molecules may include different base compositions depending on the Cas enzyme used for RNP1 and RNP2. For the circular design of blocked nucleic acid molecules, the 5′ and 3′ ends are covalently linked together. This configuration makes internalization of the blocked nucleic acid molecule into RNP2— and subsequent RNP2 activation—sterically unfavorable, thereby blocking the progression of a CRISPR Cascade reaction. Thus, RNP2 activation (e.g., trans-cleavage activity) happens after cleavage of a portion of the blocked nucleic acid molecule followed by linearization and internalization of unblocked nucleic acid molecule into RNP2.

In some embodiments, the blocked nucleic acid molecules are topologically circular molecules with 5′ and 3′ portions hybridized to each other using DNA, RNA, LNA, BNA, or PNA bases which have a very high melting temperature (Tm). The high Tm causes the structure to effectively behave as a circular molecule even though the 5′ and 3′ ends are not covalently linked. The 5′ and 3′ ends can also have base non-naturally occurring modifications such as phosphorothioate bonds to provide increased stability.

In embodiments where the blocked nucleic acid molecules are circularized (e.g., circular or topologically circular), as illustrated in FIG. 2H, each blocked nucleic acid molecule includes a first region, which is a target sequence specific to the gRNA of RNP2, and a second region, which is a sequence that can be cleaved by nuclease enzymes of activated RNP1 and/or RNP2. The first region may include a nuclease-resistant nucleic acid sequence such as, for example, a phosphorothioate group or other non-naturally occurring nuclease-resistant base modifications, for protection from trans-endonuclease activity. In some embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence. In other embodiments, when the Cas enzyme in RNP1 is Cas12a and the Cas enzyme in RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In yet other embodiments, when the Cas enzyme in RNP1 is Cas13a and the Cas enzyme in RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In some other embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable RNA sequence.

The Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acids described above may also be blocked primer molecules. Blocked primer molecules include a sequence complementary to a primer binding domain (PBD) on a template molecule (see description below in reference to FIGS. 3A and 3B) and can have the same general structures as the blocked nucleic acid molecules described above. A PBD serves as a nucleotide sequence for primer hybridization followed by primer polymerization by a polymerase. In any of Formulas I, II, or III described above, the blocked primer nucleic acid molecule may include a sequence complementary to the PBD on the 5′ end of T. The unblocked primer nucleic acid molecule can bind to a template molecule at the PBD and copy the template molecule via polymerization by a polymerase.

Other specific embodiments of the cascade assay that utilize blocked primer molecules and are depicted in FIGS. 3A and 3B. In the embodiments using blocked nucleic acid molecules described above, activation of RNP1 and trans-cleavage of the blocked nucleic acid molecules were used to activate RNP2— that is, the unblocked nucleic acid molecules are a target sequence for the gRNA in RNP2. In contrast, in the embodiments using blocked primers, activation of RNP1 and trans-cleavage unblocks a blocked primer molecule that is then used to prime a template molecule for extension by a polymerase, thereby synthesizing activating molecules that are the target sequence for the gRNA in RNP2.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules. At left of FIG. 3A is a cascade assay reaction mix comprising 1) RNP1 s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) linear template molecules (330) (which is the non-target strand); 4) a circular blocked primer molecule (334) (i.e., a high K_d molecule); and 5) a polymerase (338), such as a 129 polymerase. The linear template molecule (330) (non-target strand) comprises a PAM sequence (331), a primer binding domain (PBD) (332) and, optionally, a nucleoside modification (333) to protect the linear template molecule (330) from 3′ →5′ exonuclease activity. Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the linear template molecule (330).

Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) combines with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334) (i.e., a high K_d molecule, where high K_d relates to binding to RNP2) upon cleavage becomes an unblocked linear primer molecule (344) (a low K_d molecule, where low K_d related to binding to RNP2), which has a region (336) complementary to the PBD (332) on the linear template molecule (330) and can bind to the linear template molecule (330).

Once the unblocked linear primer molecule (344) and the linear template molecule (330) are hybridized (i.e., hybridized at the PBD (332) of the linear template molecule (330) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the end of the unblocked primer molecule (344) and the polymerase (338) can copy the linear template molecule (330) to produce a synthesized activating molecule (346) (a complement of the non-target strand, which is a target strand). The synthesized activating molecule (346) is capable of activating RNP2 (302→308). As described above, because the nucleic acid-guided nuclease in the RNP2 (308) complex exhibits (that is, possesses) both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. As stated above in relation to blocked and unblocked nucleic acid molecules (both linear and circular), the unblocked primer molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked primer molecule, although there may be some “leakiness” where some blocked primer molecules are able to interact with the gRNA in RNP2. However, an unblocked primer molecule has a substantially higher likelihood than a blocked primer molecule to hybridize with the gRNA of RNP2.

FIG. 3A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorophore emits a fluorescent signal (313). Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating molecules (346) and triggering activation of more RNP2 (308) complexes and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4. Also, as with the cascade assay embodiment utilizing blocked nucleic acid molecules that are not blocked primers, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving blocked primer molecules and circular template molecules. The cascade assay of FIG. 3B differs from that depicted in FIG. 3A by the configuration of the template molecule. Where the template molecule in FIG. 3A was linear, in FIG. 3B the template molecule is circular. At left of FIG. 3B is a cascade assay reaction mix comprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) a circular template molecule (352) (non-target strand); 4) a circular blocked primer molecule (334); and 5) a polymerase (338), such as a 129 polymerase. The circular template molecule (352) (non-target strand) comprises a PAM sequence (331) and a primer binding domain (PBD) (332). Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the circular template molecule (352).

Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) combines with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334), upon cleavage, becomes an unblocked linear primer molecule (344), which has a region (336) complementary to the PBD (332) on the circular template molecule (352) and can hybridize with the circular template molecule (352).

Once the unblocked linear primer molecule (344) and the circular template molecule (352) are hybridized (i.e., hybridized at the PBD (332) of the circular template molecule (352) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the 3′ end of the unblocked primer molecule (344). The polymerase (338) can now use the circular template molecule (352) (non-target strand) to produce concatenated activating nucleic acid molecules (360) (which are concatenated target strands), which will be cleaved by the trans-cleavage activity of activated RNP1. The cleaved regions of the concatenated synthesized activating molecules (360) (target strand) are capable of activating the RNP2 (302→308) complex.

As described above, because the nucleic acid-guided nuclease in RNP2 (308) comprises both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. FIG. 3B at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorescent signal (313) is unquenched and can be detected. Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating nucleic acid molecules and triggering activation of more RNP2s (308) and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4. Also note that as with the other embodiments of the cascade assay, in this embodiment, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

The polymerases used in the “blocked primer molecule” embodiments serve to polymerize a reverse complement strand of the template molecule (non-target strand) to generate a synthesized activating molecule (target strand) as described above. In some embodiments, the polymerase is a DNA polymerase, such as a BST, T4, or Therminator polymerase (New England BioLabs Inc., Ipswich Mass., USA). In some embodiments, the polymerase is a Klenow fragment of a DNA polymerase. In some embodiments the polymerase is a DNA polymerase with 5′→3′ DNA polymerase activity and 3′→5′ exonuclease activity, such as a Type I, Type II, or Type III DNA polymerase. In some embodiments, the DNA polymerase, including the Phi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent® DNA polymerases (New England BioLabs Inc., Ipswich Mass., USA), or any active portion or variant thereof. Also, a 3′ to 5′ exonuclease can be separately used if the polymerase lacks this activity.

FIG. 4 depicts three mechanisms in which a cascade assay reaction can release a signal from a reporter moiety. FIG. 4 at top shows the mechanism discussed in relation to FIGS. 2A, 3A and 3B. In this embodiment, a reporter moiety 409 is a separate molecule from the blocked nucleic acid molecules present in the reaction mix. Reporter moiety (409) comprises a quencher (410) and a fluorophore (411). An activated reporter moiety (412) emits a signal from the fluorophore (411) once it has been physically separated from the quencher (410).

FIG. 4 at center shows a blocked nucleic acid molecule (403), which is also a reporter moiety. In addition to quencher (410) and fluorophore (411), a blocking moiety (407) can be seen (see also blocked nucleic acid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reporter moiety (403) comprises a quencher (410) and a fluorophore (411). In this embodiment of the cascade assay, when the blocked nucleic acid molecule (403) is unblocked due to trans-cleavage initiated by the target nucleic acid of interest binding to RNP1, the unblocked nucleic acid molecule (406) also becomes an activated reporter moiety with fluorophore (411) separated from quencher (410). Note both the blocking moiety (407) and the quencher (410) are removed. In this embodiment, reporter signal is directly generated as the blocked nucleic acid molecules become unblocked.

FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleic acid or a synthesized activation molecule at a PAM distal sequence by RNP2 generates a signal. Shown are activated RNP2 (408), unblocked nucleic acid molecule (461), quencher (410), and fluorophore (411) forming an activated RNP2 with the unblocked nucleic acid/reporter moiety intact (460). Cis-cleavage of the unblocked nucleic acid/reporter moiety (461) results in an activated RNP2 with the reporter moiety activated (462), comprising the activated RNP2 (408), the unblocked nucleic acid molecule with the reporter moiety activated (463), quencher (410) and fluorophore (411).

Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting a target nucleic acid of interest in a sample. As described above, the various embodiments of the cascade assay are notable in that, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

Target nucleic acids of interest are derived from samples. Suitable samples for testing include, but are not limited to, any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal, or bacteria. In some embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms including plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus. For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface (e.g., a nasal or buccal swab).

In some embodiments, the sample can be a viral or bacterial sample or a biological sample that has been minimally processed, e.g., only treated with a brief lysis step prior to detection. In some embodiments, minimal processing can include thermal lysis at an elevated temperature to release nucleic acids. Suitable methods are contemplated in U.S. Pat. No. 9,493,736, among other references. Common methods for cell lysis involve thermal, chemical, enzymatic, or mechanical treatment of the sample or a combination of those. In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidine isothiocyanate or guanidine HC1. Suitable methods are contemplated in U.S. Pat. Nos. 8,809,519, 7,893,251, among other references. In some embodiments, minimal processing may include contacting the sample with reducing agents such as DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleases present in the crude samples. In other embodiments, minimal processing for biofluids may include centrifuging the samples to obtain cell-debris free supernatant before applying the reagents. Suitable methods are contemplated in U.S. Pat. No. 8,809,519, among other references. In still other embodiments, minimal processing may include performing DNA/RNA extraction to get purified nucleic acids before applying CRISPR Cascade reagents.

FIG. 5A shows a lateral flow assay (LFA) device that can be used to detect the cleavage and separation of a signal from a reporter moiety. For example, the reporter moiety may be a single-stranded or double-stranded oligonucleotide with terminal biotin and fluorescein amidite (FAM) modifications; and, as described above, the reporter moiety may also be part of a blocked nucleic acid. The LFA device may include a pad with binding particles, such as gold nanoparticles functionalized with anti-FAM antibodies; a control line with a first binding moiety attached, such as avidin or streptavidin; a test line with a second binding moiety attached, such as antibodies; and an absorption pad. After completion of a cascade assay (see FIGS. 2A, 3A, and 3B), the assay reaction mix is added to the pad containing the binding particles, (e.g., antibody labeled gold nanoparticles). When the target nucleic acid of interest is present, a reporter moiety is cleaved, and when the target nucleic acid of interest is absent, the reporter is not cleaved.

A moiety on the reporter binds to the binding particles and is transported to the control line. When the target nucleic acid of interest is absent, the reporter moiety is not cleaved, and the first binding moiety binds to the reporter moiety, with the binding particles attached. When the target nucleic acid of interest is present, one portion of the cleaved reporter moiety binds to the first binding moiety, and another portion of the cleaved reporter moiety bound to the binding particles via the moiety binds to the second binding moiety. In one example, anti-FAM gold nanoparticles bind to a FAM terminus of a reporter moiety and flow sequentially towards the control line and then to the test line. For reporters that are not trans-cleaved, gold nanoparticles attach to the control line via biotin-streptavidin and result in a dark control line. In a negative test, since the reporter has not been cleaved, all gold conjugates are trapped on control line due to attachment via biotin-streptavidin. A negative test will result in a dark control line with a blank test line. In a positive test, reporter moieties have been trans-cleaved by the cascade assay, thereby separating the biotin terminus from the FAM terminus. For cleaved reporter moieties, nanoparticles are captured at the test line due to anti-FAM antibodies. This positive test results in a dark test line in addition to a dark control line.

In some embodiments, the LFA device is designed for syndromic testing. For example, multiple strips with pooled RNP1s targeting different target nucleic acids of interest may be employed, either as separate devices or in a combined device. As a non-limiting example, a syndromic testing device could include four lateral flow strips, with each strip indicating the presence of at least one out of several generally related (e.g., by genetics or by treatment) pathogens (FIG. 5B). One example of a use for syndromic testing is in respiratory illness. For example, the first lateral flow strip could indicate the presence of at least one of the several strains of influenza that cause the common flu (e.g., influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, and influenza B); the second lateral flow strip could indicate the presence of at least one of the multiple strains of respiratory syncytial virus (RSV), such as RSV-A and RSV-B; the third lateral flow strip could indicate the presence of at least one variant of SARS-CoV-2 (e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5); and the fourth lateral flow strip could indicate the presence of at least one of other pathogens of interest (e.g., parainfluenza virus 1-4, human metapneumovirus, human rhinovirus, human enterovirus, adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus 0C43, MERS, and many more). The results shown in FIG. 5B indicate a positive test for the presence of RSVA and/or RSV B nucleic acid molecules. Also as seen in FIG. 5B, the syndromic testing device could further include a lateral flow strip for a negative control and a lateral flow strip for a positive control.

The components of the cascade assay may be provided in various kits. In one aspect, the kit for detecting a target nucleic acid of interest in a sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), blocked nucleic acid molecules, and reporter moieties. The first complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid(s) of interest. Binding of the first complex (RNP1) to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The blocked nucleic acid molecule comprises a sequence complementary to the second gRNA, where trans-cleavage of the blocked nucleic acid molecule results in an unblocked nucleic acid molecule and the unblocked nucleic acid molecule can bind to the second complex (RNP2), thereby activating the trans-cleavage activity of the second nucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2 results in an exponential increase in unblocked nucleic acid molecules and in active reporter moieties, where reporter moieties are nucleic acid molecules and/or are operably linked to the blocked nucleic acid molecules and produce a detectable signal upon cleavage by RNP2.

In a second aspect, the kit for detecting a target nucleic acid molecule in sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), template molecules, blocked primer molecules, a polymerase, NTPs, and reporter moieties. The first ribonucleoprotein complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid of interest and where binding of RNP1 to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The template molecules comprise a primer binding domain (PBD) sequence as well as a sequence corresponding to a spacer sequence of the second gRNA. The blocked primer molecules comprise a sequence that is complementary to the PBD on the template nucleic acid molecule and a blocking moiety.

Upon binding to the target nucleic acid of interest, RNP1 becomes active triggering trans-cleavage activity that cuts at least one of the blocked primer molecules to produce at least one unblocked primer molecule. The unblocked primer molecule hybridizes to the PBD of one of the template nucleic acid molecules, is trimmed of excess nucleotides by the 3′-to-5′ exonuclease activity of the polymerase and is then extended by the polymerase and NTPs to form a synthesized activating molecule with a sequence that is complementary to the second gRNA of RNP2. Upon activating RNP2, additional trans-cleavage activity is initiated, cleaving at least one additional blocked primer molecule. Continued cleavage of blocked primer molecules and subsequent activation of more RNP2s proceeds at an exponential rate. A signal may is generated upon cleavage of a reporter molecule by active RNP2 complexes; therefore, a change in signal production indicates the presence of the target nucleic acid molecule.

Any of the kits described herein may further include a sample collection device, e.g., a syringe, lancet, nasal swab, or buccal swab for collecting a biological sample from a subject, and/or a sample preparation reagent, e.g., a lysis reagent. Each component of the kit may be in separate container or two or more components may be in the same container. The kit may further include a lateral flow device used for contacting the biological sample with the reaction mixture, where a signal is generated to indicate the presence or absence of the target nucleic acid molecule of interest. In addition, the kit may further include instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

Example I: Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by various methods depending on the cell type and source (e.g., tissue, blood, saliva, environmental sample, etc.). Mechanical lysis is a widely-used cell lysis method and may be used to extract nucleic acids from bacterial, yeast, plant and mammalian cells. Cells are disrupted by agitating a cell suspension with “beads” at high speeds (beads for disrupting various types of cells can be sourced from, e.g., OPS Diagnostics (Lebanon N.J., US) and MP Biomedicals (Irvine, Calif., USA)). Mechanical lysis via beads begins with harvesting cells in a tissue or liquid, where the cells are first centrifuged and pelleted. The supernatant is removed and replaced with a buffer containing detergents as well as lysozyme and protease. The cell suspension is mixed to promote breakdown of the proteins in the cells and the cell suspension then is combined with small beads (e.g., glass, steel, or ceramic beads) that are mixed (e.g., vortexed) with the cell suspension at high speeds. The beads collide with the cells, breaking open the cell membrane with shear forces. After “bead beating”, the cell suspension is centrifuged to pellet the cellular debris and beads, and the supernatant may be purified via a nucleic acid binding column (such as the MagMAXTM Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham, Mass., USA) and others from Qiagen (Hilden Germany), TakaraBio (San Jose, Calif., USA), and Biocomma (Shenzen, China)) to collect the nucleic acids (see the discussion of solid phase extraction below).

Solid phase extraction (SPE): Another method for capturing nucleic acids is through solid phase extraction. SPE involves a liquid and stationary phase, which selectively separate the target analyte (here, nucleic acids) from the liquid in which the cells are suspended based on specific hydrophobic, polar, and/or ionic properties of the target analyte in the liquid and the stationary solid matrix. Silica binding columns and their derivatives are the most commonly used SPE techniques, having a high binding affinity for DNA under alkaline conditions and increased salt concentration; thus, a highly alkaline and concentrated salt buffer is used. The nucleic acid sample is centrifuged through a column with a highly porous and high surface area silica matrix, where binding occurs via the affinity between negatively charged nucleic acids and positively charged silica material. The nucleic acids bind to the silica matrices, while the other cell components and chemicals pass through the matrix without binding. One or more wash steps typically are performed after the initial sample binding (i.e., the nucleic acids to the matrix), to further purify the bound nucleic acids, removing excess chemicals and cellular components non-specifically bound to the silica matrix. Alternative versions of SPE include reverse SPE and ion exchange SPE, and use of glass particles, cellulose matrices, and magnetic beads.

Thermal lysis: Thermal lysis involves heating a sample of mammalian cells, virions, or bacterial cells at high temperatures thereby damaging the cellular membranes by denaturizing the membrane proteins. Denaturizing the membrane proteins results in the release of intracellular DNA. Cells are generally heated above 90° C., however time and temperature may vary depending on sample volume and sample type. Once lysed, typically one or more downstream methods, such as use of nucleic acid binding columns for solid phase extraction as described above, are required to further purify the nucleic acids.

Physical lysis: Common physical lysis methods include sonication and osmotic shock. Sonication involves creating and rupturing of cavities or bubbles to release shockwaves, thereby disintegrating the cellular membranes of the cells. In the sonication process, cells are added into lysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibit proteases. The cell samples are then placed in a water bath and a sonication wand is placed directly into the sample solution. Sonication typically occurs between 20-50 kHz, causing cavities to be formed throughout the solution as a result of the ultrasonic vibrations; subsequent reduction of pressure then causes the collapse of the cavity or bubble resulting in a large amount of mechanical energy being released in the form of a shockwave that propagates through the solution and disintegrates the cellular membrane. The duration of the sonication pulses and number of pulses performed varies depending on cell type and the downstream application. After sonication, the cell suspension typically is centrifuged to pellet the cellular debris and the supernatant containing the nucleic acids may be further purified by solid phase extraction as described above.

Another form of physical lysis is osmotic shock, which is most typically used with mammalian cells. Osmotic shock involves placing cells in DI/distilled water with no salt added. Because the salt concentration is lower in the solution than in the cells, water is forced into the cell causing the cell to burst, thereby rupturing the cellular membrane. The sample is typically purified and extracted by techniques such as e.g., solid phase extraction or other techniques known to those of skill in the art.

Chemical lysis: Chemical lysis involves rupturing cellular and nuclear membranes by disrupting the hydrophobic-hydrophilic interactions in the membrane bilayers via detergents. Salts and buffers (such as, e.g., Tris-HCl pH8) are used to stabilize pH during extraction, and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors (e.g., Proteinase K) are also added to preserve the integrity of the nucleic acids and protect against degradation. Often, chemical lysis is used with enzymatic disruption methods (see below) for lysing bacterial cell walls. In addition, detergents are used to lyse and break down cellular membranes by solubilizing the lipids and membrane proteins on the surface of cells. The contents of the cells include, in addition to the desired nucleic acids, inner cellular proteins and cellular debris. Enzymes and other inhibitors are added after lysis to inactivate nucleases that may degrade the nucleic acids. Proteinase K is commonly added after lysis, destroying DNase and RNase enzymes capable of degrading the nucleic acids. After treatment with enzymes, the sample is centrifuged, pelleting cellular debris, while the nucleic acids remain in the solution. The nucleic acids may be further purified as described above.

Another form of chemical lysis is the widely-used procedure of phenol-chloroform extraction. Phenol-chloroform extraction involves the ability for nucleic acids to remain soluble in an aqueous solution in an acidic environment, while the proteins and cellular debris can be pelleted down via centrifugation. Phenol and chloroform ensure a clear separation of the aqueous and organic (debris) phases. For DNA, a pH of 7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

Enzymatic lysis: Enzymatic disruption methods are commonly combined with other lysis methods such as those described above to disrupt cellular walls (bacteria and plants) and membranes. Enzymes such as lysozyme, lysostaphin, zymolase, and protease are often used in combination with other techniques such as physical and chemical lysis. For example, one can use cellulase to disrupt plant cell walls, lysosomes to disrupt bacterial cell walls and zymolase to disrupt yeast cell walls.

Example II: RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein was incubated with 375 nM of a target specific gRNA in 1×Buffer (10 mM Tris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl₂ at 25° C. for 20 minutes. The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease to gRNAs were tested, including 1:1, 1:2 and 1:5. The incubation temperature can range from 20° C.-37° C., and the incubation time can range from 10 minutes to 4 hours.

Example III: Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blocked nucleic acids, 2.504 of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to 37° C. at 0.015° C./second to form the desired secondary structure.

Snap cooling: For formation of the secondary structure of blocked nucleic acids, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by removing the heat source to form the desired secondary structure.

Snap cooling on ice: For formation of the secondary structure of blocked nucleic acids, 2.504 of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by placing the reaction tube on ice to form the desired secondary structure.

Example IV: Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-stranded DNA oligonucleotides 5-10 bases in length (e.g., with sequences of TTATT, TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and a quencher attached on the 5′ and 3′ ends, respectively. In one example using a Cas12a cascade, the fluorophore was FAM-6, and the quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville, Iowa). In another example using a Cas13 cascade, the reporter moieties were single stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n, r(UUAUU)n, r(A)n).

Example V: Cascade Assay

9+1 Format (final reaction mix components added at the same time): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the Methicillin resistant Staphylococcus aureus (MRSA) DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. Thereafter, the final reaction was carried out in 1×Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl_{2, 4} mM NaCl, 15 nM LbCas12a: 22.5 nM gRNA RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV) in a total volume of 9 μL. 1 μL of MRSA DNA target (with samples having as low as three copies and as many as 30000 copies—see FIGS. 6-14) was added to make a final volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

2+1+7 Format (RNP1 and MRSA target pre-incubated before addition to final reaction mix): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to RNP formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1×Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV) in a total volume of 9 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

2+1+6+1 Format (RNP1 and MRSA target pre-incubated before addition to final reaction mix and blocked nucleic acid molecule added to final reaction mix last): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1×Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, and 20 nM LbCas12a: 35 nM gRNA RNP2 in a total volume of 9 μL. Once the reaction mix was made, 1 μL (50 nM) blocked nucleic acid molecule (any one of Formula I-IV) was added for a total volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Example VI: Detection of SARS-CoV-2 with the Cascade Assay in Under 10 Minutes

To detect the presence of SARS-CoV-2 in a sample and determine the sensitivity of detection with the cascade assay, titration experiments were performed using a SARS-CoV-2 gamma-inactivated virus and a synthesized positive control. To serve as the positive control for the detection system, a plasmid containing a 316 bp SARS-CoV-2 nucleocapsid gene (N-gene) was synthesized by IDT (Integrated DNA Technologies, Coralville, Iowa). The N-gene sequence was as follows.

SARS-CoV-2 N-gene Target Sequence (Positive Control; SEQ ID NO: 3):
CTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCG
GCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAAT
TCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATG
GCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAG
AGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAG
AAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTA
AAGC

For the detection of SARS-CoV-2, a gamma-inactivated virus was incubated in a buffer at 95° C. for 1 minute in order to lyse and release viral RNA, followed by reverse transcription to convert the viral RNA to cDNA. The reverse transcription primer is designed to reverse transcribe the SARS-CoV-2 N-gene. The reverse transcription primer is as follows.

Reverse Transcription Primer (SEQ ID NO: 4):
GTTTGGCCTTGTTGTTGTT

RNP1 was preassembled with a guide RNA (gRNA) sequence designed to target the N-gene of SARS-CoV-2. The guide sequence is as follows.

Guide Sequence (SEQ ID NO: 5):
UAAUUUCUACUAAGUGUAGAUUUGAACUGUUGCGACUACGUGAU

RNP2 was preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearlizing) a circularized blocked nucleic acid molecule. A circularized blocked nucleic acid molecule was designed and synthesized. The blocked nucleic acid molecule was as follows.

Blocked nucleic acid molecule (SEQ ID NO: 6):
GTT*AT*TA*AA*TG*AC*TT*CT*CATT

where the * indicate bonds that are phosphorothioate modified. The 5′ and 3′ ends were covalently linked to form a circularized molecule. The SARS-CoV-2 gamma-inactivated virus or positive control with 1700, 170, 17, or 5 total copies of N-gene DNA, or a negative control (0 copies of N-gene), were added to a reaction mixture to begin the cascade assay. The reaction mix contained the preassembled RNP1, preassembled RNP2, a blocked nucleic acid molecule in a buffer (˜pH 8) containing 4 mM MgCl₂ and 101 mM NaCl. The buffering conditions were optimized to reduce non-specific nickase activity by the RNP complexes.

The cascade assay reaction proceeded for 20 minutes at 37° C. and fluorescence from the reporter molecule was measured. In all the SARS-CoV-2 gamma-inactivated virus and positive control titrations, a significant change in fluorescence was observed after 10 and 5 minutes, relative to the negative control (see the results in FIGS. 6 and 7). For the results shown in FIG. 6, the presence of the N-gene was detected in 10 minutes or less at 37° C. The data represent 3 independent biological replicates. Data is presented as mean±s.d. ****=p<0.0001 (student t-test). For the results shown in FIG. 7, the presence of SARS-CoV-2 was detected in 10 minutes or 5 minutes at 37° C. The data represent 3 independent biological replicates. Data is presented as mean±s.d. ****=p<0.0001 (student t-test). The results indicate that the cascade assay can detect as few as 5 SARS-CoV-2 target molecules in 10 minutes or less at room temperature.

Example VII: Detection of MRSA in 5 Minutes with Cascade Assay at 37° C.

To detect the presence of Methicillin resistant Staphylococcus aureus (MRSA) and determine the sensitivity of detection with the cascade assay, titration experiments with a MRSA DNA target nucleic acid of interest were performed. The MRSA DNA sequence (NCBI Reference Sequence NC: 007793.1) is as follows.

SEQ ID NO: 7:
ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGTTGTCGGGTTTGGTATATATTTTTATG
CTTCAAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAA
AGATAGCAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATAT
AATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAAC
GAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGT
TAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAA
AGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCA
ATACAGGAACAGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGC
TAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACC
TTCGTTCCACTTAAAACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTA
CAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCACATCTATTAGGTTATGTTGG
TCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAA
AAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACG
ATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAAC
TATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATC
CACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATG
GCATGAGTAACGAAGAATATAATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGAT
TACAACTTCACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGAC
GATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACGTTA
CAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTT
TGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAA
GATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTAT
TAGCTGATTCAGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGC
ATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAA
AATATTATTTCCAAAGAAAATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATA
AAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACA
AGGAGAAACTGGCAGACAAATTGGGTGGTTTATATCATATGATAAAGATAATCCAAACATGATGATGGCT
ATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGTAAAGTGTATG
ATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGATGAATAA

Briefly, an RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA. Specifically, RNP1 was designed to target a 20 bp region of the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 8). An RNP2 was preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a circularized blocked nucleic acid molecule. The circularized blocked nucleic acid molecule was designed and synthesized (SEQ ID NO: 6): GTT*AT*TA*AA*TG*AC*TT*CT*CATT, where the * indicate bonds that are phosphorothioate modified. The 5′ and 3′ ends were covalently linked to form a circularized molecule. MRSA DNA (SEQ ID NO: 7) with 3000, 300, 30, or 3 total copies, or a negative control (e.g., 0 copies), were added to a reaction mixture to begin the cascade assay. The reaction mix contained the preassembled RNP1, preassembled RNP2, and a circularized blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The buffering conditions were optimized to reduce non-specific nickase activity by the RNP complexes. The cascade assay proceeded for 10 minutes at 37° C., and fluorescence from the reporter moiety was measured. In all titrations, a significant change in fluorescence was observed after 10 and 5 minutes, relative to the negative control (see the results in FIG. 8). The cascade assay was initiated to identify the presence of MRSA in 10 minutes or 5 minutes at 37° C. Data represent 3 independent biological replicates. Data is presented as mean±s.d. ****=p<0.0001 (student t-test). The results indicate that the cascade assay can detect as few as 3 MRSA target molecules in only 5 minutes when at 37° C.

Example VIII: Detection of MRSA in Under 10 Minutes with a Cascade Assay at 25° C.

To detect the presence of MRSA and determine the sensitivity of detection with the cascade assay, titration experiments with MRSA DNA (SEQ ID NO: 7) were performed.

Briefly, an RNP1 was preassembled with a guide RNA (gRNA) sequence designed to target MRSA DNA. Specifically, RNP1 was designed to target the following 20 bp sequence in the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 8). An RNP2 was preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a circularized blocked nucleic acid molecule. A circularized blocked nucleic acid molecule was designed and synthesized (SEQ ID NO: 6): GTT*AT*TA*AA*TG*AC*TT*CT*CATT, where the * indicate bonds that are phosphorothioate modified. The 5′ and 3′ ends were covalently linked to form a circularized molecule.

MRSA DNA (SEQ ID NO: 7) with 30000, 3000, 300, 30, or 3 total copies, or a negative control (e.g., 0 copies), was added to a reaction mixture to begin the cascade assay. The reaction mix contained the preassembled RNP1, preassembled RNP2, the circularized blocked nucleic acid molecule in a buffer (— pH 8) containing 4 mM MgCl₂ and 101 mM NaCl. The buffering conditions were optimized to reduce non-specific nickase activity by the RNP complexes. The cascade reaction proceeded for 20 minutes at 25° C., and fluorescence by the reporter molecule was measured. In all titrations, a significant change in fluorescence was observed after 10 and 5 minutes, relative to the negative control (see the results in FIG. 9), indicating that the cascade assay can detect as few as 3 MRSA target molecules in 10 minutes or less while at room temperature. The data represent 3 independent biological replicates and is presented as mean±s.d. ****=p<0.0001 (student t-test).

Example IX: Optimized Detection of MRSA in 1 Minute with the Cascade Assay at 25° C.

RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA (SEQ ID NO: 7). Specifically, RNP1 was designed to target the following 20 bp sequence in the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 8). RNP2 was preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking a blocked nucleic acid molecule. Five different double stranded and linear blocked nucleic acid molecules were designed, synthesized, and tested: molecule C5, molecule C6, molecule C7, molecule C8, and molecule C9. The nucleotide sequences of molecules C5-C9 are as follows.

C5 (SEQ ID NO: 9):
GTTATTGAGAATTATTGTCATATTATTCTAATATTATTAAGGCTTATTC
ACTGTTATTATTATAATTATTAAGCTTATT
C6 (SEQ ID NO: 10):
GTTATTGAGAAGTTATTATCATCTATTATTAATAAGTTATTGCCACTAT
TATTGTTATAATTATTAAGCTTATT
C7 (SEQIDNO: 11):
GTTATTGAGAAGTATTATTCATCTAATTATTATAAGGCCTTATTACTGT
TATTATTAATAAGCTTATT
C8 (SEQ ID NO: 12):
GTTATTGAGAAGTCTTATTATCTAATATTATTAGGCCACTGTTATTATT
ATAATAAGCTTATT
C9 (SEQ ID NO: 13):
GTTATTGAGAAGTCATTATTATCTAATAAGTTATTGCCACTGTTATTAT
TATAATAAGCTTATT

Three copies of MRSA DNA (SEQ ID NO: 7) or a negative control (e.g., 0 copies) were added to a reaction mix to begin the cascade assay. The reaction mix contained the preassembled RNP1, preassembled RNP2, and one of the five blocked nucleic acid molecules in a buffer (˜pH 8) containing 4 mM MgCl₂ and 71 mM NaCl. These buffering conditions were optimized to reduce non-specific nickase activity by the RNP complexes. Each cascade assay proceeded for 10-20 minutes at 25° C., and fluorescence by the reporter molecule was measured for each cascade assay containing C5 (see the results shown in FIG. 10, where the presence of just 3 MRSA targets was detected in 5 minutes or less at 25° C. The data represent 9 independent biological replicates and is presented as mean±s.d. ****=p<0.0001 (student t-test), molecule C6 (see the results shown in FIG. 11, where the presence of just 3 MRSA targets was detected in 5 minutes or less at 25° C. The data represent 6 independent biological replicates and is presented as mean±s.d. ****=p<0.0001 (student t-test)), molecule C7 (see the results shown in FIG. 12, where the presence of just 3 MRSA targets was detected in 5 minutes or less at 25° C. Data represent 6 independent biological replicates and is presented as mean±s.d. ****=p<0.0001 (student t-test)), molecule C8 (see the results shown in FIG. 13, where the presence of just 3 MRSA targets was detected in 5 minutes or less at 25° C. Data represent 6 independent biological replicates and is presented as mean±s.d. ****=p<0.0001 (student t-test)), and molecule C9 (see the results shown in FIG. 14, where the presence of just 3 MRSA targets was detected in 10 minutes or less at 25° C. Data represent 6 independent biological replicates and data is presented as mean±s.d. ****=p<0.0001 (student t-test)). A significant change in fluorescence is observed after 1 minute and after 5 minutes, relative to the negative control, indicating that the cascade assay can be optimized to detect as few as 3 MRSA target molecules in as little as 1 minute while at room temperature.

While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the present disclosures. Indeed, the novel methods, apparatuses, modules, instruments and systems described herein can be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods, apparatuses, modules, instruments and systems described herein can be made without departing from the spirit of the present disclosures. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the present disclosures.

Claims

1. A composition of matter comprising a ribonucleoprotein (RNP) complex and a blocked nucleic acid molecule, wherein the blocked nucleic acid molecule is represented by Formula I, wherein

Formula I in the 5′-to-3′ direction comprises: A-(B-L)J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocked nucleic acid molecule comprises a sequence complementary to a gRNA; and

wherein the ribonucleoprotein complex comprises a nucleic acid-guided nuclease and the gRNA; and wherein the nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity.

2. The composition of matter of claim 1, wherein the nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease.

3. The composition of matter of claim 1, wherein the nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

4. The composition of matter of claim 1, wherein:

(a) T of Formula I comprises at least 80% sequence complementarity to B and C; and/or

(b) D of Formula I comprises at least 80% sequence complementarity to A.

5. The composition of matter of claim 1, wherein the blocked nucleic acid molecule comprises a modified nucleoside or nucleotide.

6. The composition of matter of claim 1, wherein the blocked nucleic acid molecule does not comprise a PAM sequence.

7. The composition of matter of claim 1, wherein the blocked nucleic acid molecule comprises a PAM sequence disposed between the first and second sequences, wherein the first sequence is 5′ to the PAM sequence.

8. The composition of matter of claim 1, further comprising a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by the ribonucleoprotein complex.

9. The composition of matter of claim 8, wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.

10. The composition of matter of claim 9, wherein the detectable signal is the fluorescent signal.

11. The composition of matter of claim 9, wherein the detectable signal is the chemiluminescent signal.

12. The composition of matter of claim 9, wherein the detectable signal is the colorimetric signal.

13. The composition of matter of claim 9, wherein the detectable signal is the other optical signal.

14. The composition of matter of claim 8, wherein the reporter moiety comprises a modified nucleoside or nucleotide.

15. The composition of matter of claim 1, further comprising a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by the ribonucleoprotein complex.

16. The composition of matter of claim 15, wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.

17. The composition of matter of claim 16, wherein the detectable signal is the fluorescent signal.

18. The composition of matter of claim 17, wherein the reporter moiety is conjugated to the fluorescent signal and a quencher.

19. The composition of matter of claim 16, wherein the detectable signal is the chemiluminescent signal.

20. The composition of matter of claim 16, wherein the detectable signal is the radioactive signal.

21. The composition of matter of claim 16, wherein the detectable signal is thecolorimetric signal.

22. The composition of matter of claim 16, wherein the detectable signal is the other optical signal.

23. The reaction mixture of claim 15, wherein the reporter moiety comprises a modified nucleoside or nucleotide.

24. The composition of matter of claim 23, wherein the modified nucleoside or nucleotide comprises a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.

25. The composition of matter of claim 1, wherein the reaction mixture comprises about 1 fM to about 1 mM of the RNP2.

26. The composition of matter of claim 1, wherein a Kd of the blocked nucleic acid molecules to the RNP is about 105-fold greater or more than the Kd of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.

27. The composition of matter of claim 26, wherein a Kd of the blocked nucleic acid molecules to the RNP is about 106-fold greater or more than the Kd of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.

28. The composition of matter of claim 27, wherein a Kd of the blocked nucleic acid molecules to the RNP is about 107-fold greater or more than the Kd of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.

29. The composition of matter of claim 28, wherein a Kd of the blocked nucleic acid molecules to the RNP is about 108-fold greater or more than the Kd of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.

30. The composition of matter of claim 29, wherein a Kd of the blocked nucleic acid molecules to the RNP is about 1010-fold greater or more than the Kd of an unblocked nucleic acid molecule resulting from unblocking of the blocked nucleic acid molecules.