Abstract: Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction.
Type:
Application
Filed:
June 22, 2022
Publication date:
October 20, 2022
Applicant:
New England Biolabs, Inc.
Inventors:
G. B. Robb, Isaac B. Meek, Dianne S. Schwarz, Ezra Schildkraut
Abstract: Methods, kits and compositions, in some embodiments, may include a thermostable DNA guided Argonaute protein for example TtAgo, a thermostable single-stranded DNA binding protein (SSB) for example, ET SSB, and, optionally, a strand-displacing polymerase. A SSB may allow (a) Argonaute/guide DNA complexes to substantially enhance cleavage efficiency of single- and double-stranded DNA substrates; (b) the use of longer guide DNAs (e.g., guide DNAs that are at least 24 nucleotides in length) and/or (c) increases in the sequence specificity of Argonaute-mediated binding and cleavage reactions.
Abstract: Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may comprise ligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.
Abstract: The present disclosure relates, according to some embodiments, to methods for preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase. In some embodiments, a tailing enzyme and/or a ligase used in library preparation may be immobilized enzymes.
Type:
Application
Filed:
May 31, 2022
Publication date:
September 22, 2022
Applicant:
New England Biolabs, Inc.
Inventors:
Ming-Qun Xu, Yi Fang, Aihua Zhang, Luo Sun
Abstract: Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.
Abstract: Methods are provided that, for example, include (a) combining ssDNA containing a modified nucleotide (e.g., a ssDNA with a modified nucleotide proximate to its 5? end) with a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate a first ssDNA fragment having a 3?OH and a second ssDNA fragment having the modified nucleotide); wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio (m/m); and (b) cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, a method may comprise (a) combining (i) a ssDNA comprising a modified nucleotide (e.g., proximate to its 5? end) with (ii) a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3?OH and a second ssDNA fragment comprising the modified nucleotide) wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio and cleaving at least 95% of the ssDNA at the modified nucleotide.
Abstract: A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.
Type:
Application
Filed:
May 11, 2022
Publication date:
September 1, 2022
Applicant:
New England Biolabs, Inc.
Inventors:
Jennifer Ong, Vladimir Potapov, Kuo-Chan Hung, Haruichi Asahara, Shaorong Chong, George Tzertzinis
Abstract: The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.
Type:
Application
Filed:
July 16, 2021
Publication date:
July 28, 2022
Applicant:
New England Biolabs, Inc.
Inventors:
Mehul Ganatra, Siu-hong Chan, Christopher H. Taron, G. B. Robb
Abstract: Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction. DNA polymerase, RNA polymerase and single stranded (ss) DNA oligonucleotides are combined where each DNA oligonucleotide has one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3? end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and forming a single RNA polynucleotide, excluding the RNA promoter sequence, derived from the first and second DNA oligonucleotides.
Type:
Grant
Filed:
April 4, 2019
Date of Patent:
July 26, 2022
Assignee:
New England Biolabs, Inc.
Inventors:
G. B. Robb, Isaac B. Meek, Dianne S. Schwarz, Ezra Schildkraut
Abstract: Compositions are provided for 3? adapters and methods of use are provided that include methods requiring a plurality of ligation steps involving a single-stranded target polynucleotide and 3? and 5? adapters. Embodiments of the 3? adapters comprise a cleavable linker positioned between a single-stranded region and a double-stranded region. Upon ligating the 3? adapters, the single-stranded region is released by cleaving the cleavable linker.
Abstract: Provided herein is a thermolabile proteinase and methods of using the same. In some embodiments, the thermolabile proteinase may comprise an amino acid sequence that is at least 90% identical to any of SEQ ID NOs:1-11 and at least one amino acid substitution in helix 3. The thermolabile proteinase is active at a temperature in the range of 4° C.-40° C. and is inactivated by raising the temperature to above 50° C., where the proteinase is substantially inactive at 65° C.
Type:
Grant
Filed:
December 21, 2020
Date of Patent:
July 19, 2022
Assignee:
New England Biolabs, Inc.
Inventors:
Minyong Chen, James C. Samuelson, Ming-Qun Xu, Aihua Zhang, Margaret Heider, Pingfang Liu
Abstract: Kits and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The kits and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The kits and methods utilize in various combinations, reversible inhibitors of kit components, thermolabile enzymes, poloxamers, various salts, indicators and one or more Loop-Mediated Isothermal Amplification (LAMP) primer sets for detecting single and/or multiple targets and variants of the targets including SARS-CoV-2 targets and variants thereof in a single reaction. The kits and methods permit detection of the target nucleic with similar sensitivity regardless of the presence of undefined mutations that may enhance the virulence of cells or viruses containing the undefined mutations.
Type:
Application
Filed:
March 21, 2022
Publication date:
July 7, 2022
Applicant:
New England Biolabs, Inc.
Inventors:
Nathan Tanner, Yinhua Zhang, Eric Hunt, Gregory Patton, Guoping Ren, Zhiru Li, Andrew Barry, Nicole Nichols, Catherine B. Poole, Harriet M. Strimpel, Ivan R. Correa, JR., Clotilde Carlow, Esta Slayton
Abstract: Compositions and methods of use are provided that among other things, allow for efficient adapter ligation to small RNAs. Embodiments of the compositions include partially double stranded polynucleotides for use as 3? adapters that contain a cleavable linker positioned between a single-stranded region and a double-stranded region. Upon ligating the 3? adapters, the single-stranded region is released by cleaving the cleavable linker.
Abstract: Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.
Abstract: The present disclosure relates, according to some embodiments, to methods for preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase. In some embodiments, a tailing enzyme and/or a ligase used in library preparation may be immobilized enzymes.
Type:
Grant
Filed:
September 11, 2020
Date of Patent:
July 5, 2022
Assignee:
New England Biolabs, Inc.
Inventors:
Ming-Qun Xu, Yi Fang, Aihua Zhang, Luo Sun
Abstract: Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may comprise ligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.
Abstract: Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.
Type:
Grant
Filed:
January 25, 2021
Date of Patent:
June 28, 2022
Assignee:
New England Biolabs, Inc.
Inventors:
Jennifer Ong, Nathan Tanner, Yinhua Zhang, Yanxia Bei, Vladimir Potapov
Abstract: Provided herein is a method for chemically capping polynucleotides having a 5? monophosphate. In some embodiments the method may comprise: combining an activated nucleoside 5? mono- or poly-phosphate with a population of polynucleotides that comprises polynucleotides having a 5? monophosphate, to produce a reaction mix; and incubating the reaction mix to produce reaction products that comprise a polynucleotide and a 5? nucleoside cap, linked by a 5? to 5? polyphosphate linkage. The chemical capping method described herein can be incorporated into a variety of cDNA synthesis methods.
Type:
Application
Filed:
May 6, 2020
Publication date:
June 23, 2022
Applicant:
New England Biolabs, Inc.
Inventors:
Ivan R. Correa, Jr., Shengxi Guan, Madalee G. Wulf, Nan Dai, Sean Maguire
Abstract: A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.
Type:
Grant
Filed:
November 11, 2019
Date of Patent:
June 14, 2022
Assignee:
New England Biolabs, Inc.
Inventors:
Jennifer Ong, Vladimir Potapov, Kuo-Chan Hung, Haruichi Asahara, Shaorong Chong, George Tzertzinis
Abstract: Ordered assembly of large numbers of fragments into a single large DNA have been improved in both frequency and fidelity of the assembled product. This has been achieved by novel compositions and methods that are utilized in a computer system that integrates comprehensive ligation data from multiple sources to provide optimized synthetic overhangs or overhangs from restriction endonuclease cleavage on DNA fragments for assembly by ligation. Intragenic cut sites are avoided by the use of a novel restriction endonuclease which recognizes 7 nucleotides (bases) and cuts DNA to create 4-base overhangs with the help of a synthetic activator oligonucleotide. Variations in ligation preferences by different ligases provide extra precision in assembly reactions. The use of the improved methods are exemplified by the successful assembly from 52 fragments of a viral genome and also a 52 fragment ordered assembly of a bacteria operon.
Type:
Application
Filed:
December 17, 2021
Publication date:
June 9, 2022
Applicant:
New England Biolabs, Inc.
Inventors:
Gregory Lohman, Vladimir Potapov, John M. Pryor, Rebecca Kucera, Katharina Bilotti, Richard D. Morgan