ENZYMES
The invention relates to a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a DNA nucleotide template, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at one or more residues of the thumb region, said residues selected from: amino acids 651 to 679 (patch 10A); wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at residue E664. In one embodiment said polymerase comprises the mutations Y409G and E664K. In one embodiment said polymerase comprises amino acid sequence corresponding to SEQ ID NO:12. The invention also relates to A nucleic acid polymerase capable of reverse transcribing a HNA nucleotide polymer into a DNA nucleotide polymer, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of sEQ ID NO:1, wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at residue I521.
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This application is a continuation of U.S. patent application Ser. No. 14/872,814, filed Oct. 1, 2015, which is a continuation of U.S. patent application Ser. No. 13/695,377, filed Oct. 30, 2012 (now U.S. Pat. No. 9,169,471, issued on Oct. 27, 2015), which is a national stage entry of PCT/GB2011/000583, filed Apr. 14, 2011, which claims priority benefit of British Application No. 1007384.9, filed Apr. 30, 2010, and U.S. Provisional Application No. 61/396,008, filed May 20, 2010, the contents of which are hereby incorporated by reference in their entireties.
SEQUENCE LISTINGThis Application incorporates by reference an ASCII text file Sequence Listing named U.S. Ser. No. 14/872,814.txt, created Mar. 18, 2018, and containing 241,753 bytes.
BACKGROUNDLife's diversity is largely based on the versatility of two polymers: polypeptides (i.e., proteins) and polynucleotides (nucleic acids). Information storage and propagation in biological systems is commonly based on just two types of nucleic acids, DNA and RNA. Nucleic acids in particular display unique properties beyond their ability to encode genetic information, which make them important tools in chemistry, biotechnology, nanotechnology and medicine. Nucleic acids also have enormous potential as therapeutics but suffer from systemic constraints inherent in DNA and RNA chemistry such as poor serum/nuclease stability.
Life's diversity is largely based on the versatility of two polymers: polypeptides (i.e., proteins) and polynucleotides (nucleic acids). Information storage and propagation in biological systems is commonly based on just two types of nucleic acids, DNA and RNA. Nucleic acids in particular display unique properties beyond their ability to encode genetic information, which make them important tools in chemistry, biotechnology, nanotechnology and medicine. Nucleic acids also have enormous potential as therapeutics but suffer from systemic constraints inherent in DNA and RNA chemistry such as poor serum/nuclease stability.
Systematic chemical studies have begun to uncover the critical chemical and physico-chemical parameters that have enabled DNA and RNA to serve as the molecular basis for life's genetic systems. Changes to the chemical structure of nucleic acids, including unnatural nucleobases have been used to investigate the molecular determinants for information storage. Synthetic exploration of alternative backbone linkages and ribofuranose congeners have been explored and have revealed the profound influence of the chemical makeup of backbone and/or sugar (or equivalent) chemistry on nucleic acid properties, structure and conformation. Crucially, only a small subset of chemistries allows cross-polymer information transfer through efficient pairing with DNA or RNA: a prerequisite for the formation of a synthetic genetic system capable of crosstalk with extant biology. However, cross-hybridization experiments alone cannot conclusively determine the capacity of a given chemistry to serve as a genetic system as hybridization does not necessarily preserve information content.
A more thorough examination of the potential of a potential genetic polymer for information storage, propagation and evolution requires a system of replication. In principle, artificial polymers might be synthesized and replicated chemically but non-enzymatic polymerization is usually inefficient and error-prone and consequently unattractive as a generic approach despite significant advances in the polymerization of mononucleotide or short oligomer (pentamer) units using specialized chemistries. Enzymatic polymerization using DNA or RNA polymerases is potentially powerful but is restricted but the tight substrate specificity of natural polymerases. Despite significant progress in understanding determinants of polymerase substrate specificity and the engineering of polymerases with expanded substrate spectra, most unnatural nucleotide analogues have remained inadequate polymerase substrates at full substitution for either synthesis and/or as templates for reverse transcription.
DNA and RNA are not only a repository of genetic information for life. They are also unique polymers with remarkable properties: they associate according to well-defined rules, can be assembled into diverse nanostructures of defined geometry, can be evolved to bind ligands and catalyze chemical reactions and can serve as a supramolecular scaffold to arrange chemical groups in space.
Aptamers are a promising class of biomolecular therapeutics based on structured single-stranded nucleic acids with the potential to rival antibodies in some clinical settings. A broad spectrum of both RNA- and DNA-based aptamers have been described directed against a wide-range of targets and several are currently undergoing in clinical trials underlining their potential. Reagents based on natural nucleic acids such as RNA or DNA have drawbacks, however, with respect to a number of desirable properties for clinical reagents and therapeutics, such as in vivo stability and/or bioavailability. In principle, aptamers may be stabilized (post-selection) by medicinal chemistry approaches and this approach has been validated by Macugen, the 1st aptamers based drug, which has been approved for the treatment of macular degeneration. However, post-selection modifications can alter and/or weaken aptamer structure and target interactions and may modify aptamer specificity, which is a problem.
A wide range of modified nucleotides has been used in SELEX to create aptamers comprising unnatural chemistries. Some of these modifications confer desirable characteristics on the selected aptamers such as increased nuclease resistance and stability but also have drawbacks such as toxicity and increased non-specific interaction with proteins.
Orthogonality (i.e., a lack of interaction/interference with the cellular machinery) and the resulting lack of toxicity, increased nuclease resistance as well as other potentially desirable properties may in principle arise from the use of more radically engineered nucleic acids. Their application to the aptamer field, however, requires both the design and synthesis of such nucleic acids as well as the generation of custom-made polymerases for their synthesis, replication and evolution. It is a problem that such reagents and polymerases do not exist in the art.
Many novel nucleic acid structures have been built with a view towards increased orthogonality. The challenge here is to design scaffolds that lead to minimal interaction/interference with the cellular genetic machinery while simultaneously maintaining an ability to communicate with it. Notable achievements include attempts at expanding the genetic alphabet (informational orthogonality) and altering the structure or size of nucleobases (steric orthogonality). In each of these cases, however, issues of cellular toxicity and/or informational specificity remain.
A different approach towards chemically orthogonal nucleic acids involves the modification of the backbone but leaves the informational nucleobases intact. Replacement of the canonical ribofuranose with other pentoses (or hexoses and tetroses) can indeed have dramatic effects on helical conformation and duplex stability and formation.
SUMMARYCertain orthogonal nucleic acids are known in the art. These are based on orthogonal nucleic acid chemistry so as to be structurally different from naturally occurring DNA or RNA. One example of an orthogonal nucleic acid is that based on hexitol (HNA). Another example is one based on cyclohexenyl (CeNA) nucleotides. To date, however, such orthogonal nucleic acids have only been able to be produced chemically. This has meant that production of a polymer of biologically useful length has been extremely demanding, and expensive.
The present invention provides nucleic acid polymerases that are able to polymerise orthogonal nucleotides into orthogonal nucleic acid polymers. The inventors have created and selected individual polymerase enzymes possessing orthogonal polymerase activities. As a result of their studies, the inventors have defined specific regions and patches in the polymerases which determine and allow the manipulation of the polymerase activity towards the orthogonal nucleotides. The invention is based upon these surprising findings.
Thus in one aspect the invention provides nucleic acid polymerases capable of producing a non-DNA nucleotide polymer from a DNA nucleotide polymer template, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at one or more residues of the thumb region, said residues selected from: amino acids 651 to 679 (patch 10A); wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at residue E664.
Suitably, said non-DNA nucleotide polymer is a RNA polymer; and said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at residue Y409. Suitably, when the non-DNA nucleotide polymer is a RNA polymer then the polymerase comprises the mutation Y409N or Y409G. Suitably, when the non-DNA nucleotide polymer is a RNA polymer then the polymerase comprises the mutation E664K or E664Q. Suitably, when the non-DNA nucleotide polymer is a RNA polymer then the polymerase comprises the mutations Y409N and E664Q (TgoT Y409N E664Q; TNQ). Suitably, when the non-DNA nucleotide polymer is a RNA polymer then the polymerase comprises the mutations Y409G and E664K (TgoT Y409G E664K; TGK). Suitably, said polymerase comprises the mutations Y409G and E664K. In another aspect, the invention relates to a method for making a RNA nucleotide polymer, said method comprising contacting a DNA template with a nucleic acid polymerase as described above and incubating to allow polymerisation.
In another aspect, the invention relates to a nucleic acid polymerase as described above wherein said polymerase is capable of producing a HNA nucleotide polymer from a DNA nucleotide polymer template and said polymerase comprises amino acid sequence corresponding to amino acids 651 to 679 (patch 10A) of SEQ ID NO:12. Suitably, said polymerase is capable of producing a HNA nucleotide polymer from a DNA nucleotide polymer template and said polymerase comprises amino acid sequence corresponding to SEQ ID NO:12. In another aspect, the invention relates to a method for making a HNA nucleotide polymer, said method comprising contacting a DNA template with a nucleic acid polymerase as described above and incubating to allow polymerisation.
In another aspect, the invention relates to a nucleic acid polymerase capable of reverse transcribing a HNA nucleotide polymer into a DNA nucleotide polymer, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at residue I521. Suitably, said polymerase comprises a mutation selected from the group consisting of: I521L, I521P and I521H. Suitably said polymerase comprises the mutation I521L.
Suitably, said polymerase further comprises the mutation A485L.
Suitably, said polymerase further comprises the mutation V93Q.
Suitably, said polymerase further comprises the mutations E141A and E143A.
In another aspect, the invention relates to a nucleic acid polymerase as described above wherein said non-DNA polymer is a HNA polymer and wherein said polymerase comprises the mutation I521L and said polymerase further comprises the mutation A485L and said polymerase further comprises the mutation V93Q and said polymerase further comprises the mutations E141A and E143A.
In another aspect, the invention relates to a method for making a DNA nucleotide polymer, said method comprising contacting a HNA template with a nucleic acid polymerase as described above and incubating to allow polymerisation.
In another aspect, the invention relates to a nucleic acid encoding a polymerase as described above.
In another aspect, the invention relates to a host cell comprising a nucleic acid as described above.
In another aspect, the invention relates to a system comprising: (i) a nucleic acid polymerase capable of producing a HNA nucleotide polymer from a DNA nucleotide polymer template; and (ii) a nucleic acid polymerase capable of reverse transcribing a HNA nucleotide polymer into a DNA nucleotide polymer.
In another aspect the invention provides a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a DNA nucleotide polymer template, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at one or more residues of the thumb region, said residues selected from: (i) amino acids 651 to 679 (patch 10A); or (ii) amino acids 734 to 765 (patch 12).
Suitably, said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at one or more residues selected from: (i) amino acids 651 to 679 (patch 10A).
Suitably, said polymerase is capable of producing a HNA nucleotide polymer from a DNA nucleotide polymer template, and said polymerase comprises amino acid sequence corresponding to amino acids 651 to 679 (patch 10A) of any of SEQ ID NO:12, NO:13, NO:14, or NO:15; and/or said polymerase comprises amino acid sequence corresponding to amino acids 734 to 765 (patch 12) of any of SEQ ID NO:16, NO:17, or NO:18.
Suitably, said polymerase is capable of producing a HNA nucleotide polymer from a DNA nucleotide polymer template and said polymerase comprises amino acid sequence corresponding to SEQ ID NO:12.
Suitably, said polymerase is capable of producing a CeNA nucleotide polymer from a DNA nucleotide polymer template and said polymerase comprises amino acid sequence corresponding to amino acids 651 to 679 (patch 10A) of any of SEQ ID NO:3, NO:4, NO:5, NO:6, NO:7, NO:8, NO:9 or NO:10; and/or said polymerase comprises amino acid sequence corresponding to amino acids 734 to 765 (patch 12) of any of SEQ ID NO:11.
Suitably, said polymerase is capable of producing a CeNA nucleotide polymer from a DNA nucleotide polymer template and said polymerase comprises amino acid sequence corresponding to SEQ ID NO:5 or SEQ ID NO:6.
Suitably, said polymerase comprises the mutation E664Q.
Suitably, said polymerase is capable of producing a RNA nucleotide polymer from a DNA nucleotide polymer template and said polymerase comprises the mutation E664Q.
Suitably, said polymerase further comprises the mutation A485L.
Suitably, said polymerase further comprises the mutation V93Q.
Suitably, said polymerase further comprises the mutations E141A and E143A.
In another aspect, the invention relates to a method for making a non-DNA nucleotide polymer, said method comprising contacting a DNA template with a nucleic acid polymerase as described above and incubating to allow polymerisation. Suitably, synthesis may be followed by suitable step(s) to either dissociate said polymer from the DNA template or remove the DNA template to free the polymer.
In another aspect, the invention relates to a non-DNA nucleotide polymer which comprises at least 50 nucleotides. Suitably, said non-DNA nucleotide polymer comprises HNA or CeNA.
In another aspect, the invention relates to a non-DNA nucleotide polymer obtained by the method as described above.
In another aspect, the invention relates to a nucleic acid polymerase capable of reverse transcribing a non-DNA nucleotide polymer into a DNA nucleotide polymer, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at one or more residues selected from: I521 and Y388.
Suitably, said non-DNA polymer is an HNA polymer or a CeNA polymer or a RNA polymer.
Suitably, said polymerase capable of reverse transcription comprises a mutation selected from the group consisting of: I521L, I521P and I521H. Suitably, said polymerase comprises the mutation I521L.
Suitably, said polymerase capable of reverse transcription comprises a mutation selected from the group consisting of: Y388V, Y388R, Y388H, Y388N and Y388T.
Suitably, said polymerase capable of reverse transcription further comprises the mutation A485L.
Suitably, said polymerase capable of reverse transcription further comprises the mutation V93Q.
Suitably, said polymerase capable of reverse transcription further comprises the mutations E141A and E143A.
Suitably, said non-DNA polymer is a HNA polymer and said polymerase capable of reverse transcription comprises the mutation I521L and said polymerase further comprises the mutation A485L and said polymerase further comprises the mutation V93Q and said polymerase further comprises the mutations E141A and E143A.
In another aspect, the invention relates to a nucleic acid encoding a polymerase as described above.
In another aspect, the invention relates to a host cell comprising a nucleic acid as described above.
In another aspect, the invention relates to a method of screening for a non-DNA nucleotide polymer having a particular predetermined characteristic, said method comprising preparing a candidate non-DNA nucleotide polymer as described above, and assaying said non-DNA nucleotide polymer for said characteristic.
In another aspect, the invention relates to a method for making a DNA polymer from a non-DNA nucleotide polymer template, said method comprising contacting said template with a nucleic acid polymerase as described above and incubating to allow polymerisation.
In another aspect, the invention relates to a method of making a medicament, said method comprising preparing a non-DNA nucleotide polymer as described above.
In another aspect, the invention relates to a system comprising (i) a nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a DNA nucleotide polymer template as described above, and (ii) a nucleic acid polymerase capable of reverse transcribing a non-DNA nucleotide polymer into a DNA nucleotide polymer as described above.
In another aspect, the invention relates to a system comprising (i) a nucleic acid polymerase capable of producing a repertoire of diverse non-DNA nucleotide polymers from a repertoire of diverse DNA nucleotide polymer templates as described above; (ii) the selection or screening of above repertoire of diverse non-DNA nucleotide polymers for a desired function such as (1) binding to a specified target, or (2) the catalysis of a specified chemical reaction; and (iii) a nucleic acid polymerase capable of reverse transcribing the selected non-DNA nucleotide polymer with the desired phenotype (binding or catalysis) into a DNA nucleotide polymer as described above.
Information storage and propagation in biology is based on just two types of nucleic acids, DNA and RNA. It is not known, however, whether their prevalence was the result of circumstance (“historic accident”), reflecting constraints imposed at the origin of life, or if chemical alternatives to ribofuranose-based nucleic acids are limited. Here, we describe the development of an artificial genetic system based on 1,5 anhydrohexitol nucleic acids (HNA), a chemically simple nucleic acid architecture not found in nature, and demonstrate this system's capacity for heredity and evolution. Using polymerase evolution and design, we have engineered both a DNA-templated HNA polymerase and an HNA reverse transcriptase. Together these allow efficient transfer of genetic information from DNA into HNA and back into DNA, establishing HNA-based heredity with an aggregate fidelity of 1.4×10−2—comparable to RNA virus replication. We furthermore demonstrate evolution of this new synthetic genetic material by the de novo selection of specific all-HNA aptamers against an RNA target (HIV-TAR). Our results show that both heredity and evolution can be provided by an entirely unnatural system, suggesting that there is no fundamental functional imperative for life to be built on ribofuranose-based nucleic acids.
The present invention relates to the design and construction of an artificial genetic system based on a third type of genetic material using orthogonal nucleic acid chemistry and in particular evolved polymerases for the templated synthesis, replication and evolution of novel, sequence-defined nucleic acid polymers.
Such a system is a key enabling technology for the synthesis, replication and evolution of novel nucleic acid polymers with potential applications as diagnostics, prognostics and therapeutics. It also promises to address some of the systemic constraints inherent in DNA and RNA chemistry with regards to therapeutic application.
We have selected two unnatural nucleic acid architectures, Hexitol nucleic acid (HNA) and Cyclohexenyl nucleic acids (CeNA) as exemplary backbone structures to illustrate the invention. HNA and CeNA nucleotides are very poor substrates for naturally occurring kinases and are thus non-toxic to cells. HNA and CeNA polymers are not substrates for naturally occurring nucleases and are therefore not degraded by either DNases or RNAses. Both HNA and CeNA are completely resistant to nuclease degradation and appear not to be substrates for DNA or RNA modifying enzymes. Significantly, they are non-toxic to cells as nucleotides and appear to be not recognized as substrates by the cellular replication, transcription and translation machinery. At the same time, they crucially retain the ability to form sequence specific duplexes with DNA and RNA, an essential property for the transfer of genetic information. HNA is known (and CeNA is predicted) to form duplex structures that are different from canonical A- or B-form helices, which may allow formation of novel aptamer conformational and structural motifs with potential for novel functions. HNA monomers (such as might arise from the eventual degradation of long-lived therapeutics) have been shown to be non-toxic to cells. HNA and CeNA therefore unite an ideal spectrum of properties for the assembly of an orthogonal genetic system and its application to the isolation of nucleic acid therapeutics such as aptamers.
Suitably, for HNA polymerase applications, the polymerase of the invention comprises mutations as shown for ‘6G12’.
Suitably, for RNA polymerase applications, the polymerase of the invention comprises mutations as shown for ‘D4’ such as ‘D4N3’. In particular this comprises a Y409 mutation which in ‘D4N3’ is Y409N.
Suitably, for CeNA polymerase applications of the invention, the polymerase comprises mutations as shown for ‘C7’ or 6G12.
It will be appreciated that numerous polymerases of the invention show activity for multiple orthogonal nucleic acids. For example, one enzyme can display both RNA reverse transcriptase and HNA reverse transcriptase activity. If that enzyme has the proof reading function intact then it is unlikely to be suitable as an HNA reverse transcriptase, but remains suitable as an RNA reverse transcriptase.
To be considered a useful enzyme according to the invention (i.e., to be considered capable of having the specified functions, the polymerase or reverse transcriptase of the invention should be able to produce a polymer of at least 14 nucleotides in length, suitably at least 15 nucleotides in length; more suitably 40 nucleotides in length, most suitably at least 50 nucleotides in length.
According to the most stringent criterion of synthesis of a polymer at least 50 nucleotides in length, polymerases C7 and 6G12 may not be regarded as acting on RNA; polymerases D4 and C7 may not be regarded as acting on HNA. Thus, if polymerases of the invention are discussed as being for or specific for a particular type of orthogonal nucleic acid, it should be understood that they are expected to be able to consistently produce a polymer or at least 40 nucleotides, suitably at least 50 nucleotides in length.
Typically, the smallest aptamers or ribozymes need approximately 40 nucleotides of sequence in order to fold. More suitably, small aptamers or ribozymes also comprise a few extra nucleotides for polymerase binding, therefore suitably being at least about 50 nucleotides in length. For aptamer screening applications, a typical preferred minimum length is therefore 50 nucleotides.
DefinitionsThe term “comprises” (comprise, comprising) should be understood to have its normal meaning in the art, i.e., that the stated feature or group of features is included, but that the term does not exclude any other stated feature or group of features from also being present.
As used herein, the term reverse transcriptase or “reverse transcriptase activity” refers to the manufacture of a DNA polymer from a non DNA polymer template. Thus, when the non DNA template comprises RNA, then the term has its normal meaning in the art. As will be apparent from the context, in numerous aspects and embodiments the invention relates to reverse transcriptase activity in the sense of manufacturing a DNA polymer from a non DNA polymer template which may be, for example, RNA, HNA, CeNA, or other such non DNA template. Thus, reverse transcriptase may be classically regarded as an RNA dependant DNA polymerase. For other non DNA nucleotide polymers the term “reverse transcriptase” has the same overall meaning, except that the template nucleic acid may of course vary. For example, a reverse transcriptase for HNA means an HNA dependant DNA polymerase; a reverse transcriptase for CeNA means a CeNA dependant DNA polymerase.
A non DNA nucleotide means a nucleotide other than a deoxy ribonucleotide. For example, it may mean a conventional ribonucleotide which may be used to make an RNA polymer. Alternatively, it may mean any other kind of non DNA nucleotide, such as a hexitol (HNA) nucleotide or a cyclohexenyl (CeNA) nucleotide or other such nucleotide polymer. Most suitable non DNA nucleotide or polymer is also a non RNA nucleotide or polymer, i.e., most suitably a non DNA polymer may be XNA or 3NA (such as HNA or CeNA).
Suitably, the invention relates to the production of a complete new polymer, i.e., orthogonal nucleic acid. The aim is to keep the information which is encoded in that polymer the same as the information encoded in a conventional DNA polymer. For example, it is not an aim of the invention to include a fifth base or to expand the genetic alphabet beyond the four conventional bases, although the invention could be applied in this manner by the skilled operator if desired. Suitably, the polymers of the invention reflect the same four bases as conventional DNA polymers in terms of their information content.
Orthogonal nucleic acids are non DNA nucleic acids. Examples include HNA or CeNA as described herein. These non DNA nucleic acids are sometimes referred to as “3NA” or as “XNA” as will be apparent from the context. Suitably, the orthogonal nucleic acid is HNA or CeNA. Most suitably, the orthogonal nucleic acid is HNA. HNA is particularly advantageous since it is the most experimentally tractable of the orthogonal nucleic acids discussed herein.
PolymeraseIn principle, polymerases of the invention may be made by introducing the specific mutations described herein into the corresponding site of a starting polymerase or “polymerase backbone” of the operator's choice. In this way, the activity of that starting polymerase may be modified to provide the orthogonal activities as described herein.
The polymerase backbone may be any member of the well-known polB enzyme family (including the pol delta variant which shows only 36% identity with the exemplary true wild type TgoT sequence of SEQ ID NO:1). More suitably, the polymerase backbone may be any member of the well-known polB enzyme family excluding viral polymerases. More suitably, the polymerase backbone may be any member of the well-known polB enzyme family having at least 36% identity to SEQ ID NO:1; suitably at least 50%; suitably at least 60%; suitably at least 70%; suitably at least 80%. At the 80% identity level, the invention suitably embraces polB enzymes from the Archaeal, Thermococcus and/or Pyrococcus genera. In a preferred embodiment, suitably, the polymerase backbone has at least 90% identity to SEQ ID NO:1.
When using other polymerase backbones, mutations are transferred to the equivalent position as is well known in the art and as noted above. For example, with reference to the exemplary polymerase 6G12, the following table illustrates how the transfer of mutations to alternate backbones may be carried out. Table 1 shows Pol6G12 mutations and structural equivalent positions in other PolBs. The mutations found in Pol6G12 are shown against the underlying sequence of the wild-type Tgo. The structurally equivalent residue in other well-studied B-family polymerases is given. Residues that were not mapped to equivalent positions are shown as N.D.
Most suitably, the polymerase backbone is Archaeal thermococcus TgoT polB; the true wild type sequence is as shown in SEQ ID NO:1. This will serve as the reference sequence, and preferred embodiments of the invention are described with reference to this sequence.
Reference SequenceWhen particular amino acid residues of polymerase are referred to using numeric addresses, the numbering is taken with reference to the true wild type TgoT polB amino acid sequence of SEQ ID NO:1 (or to the nucleic acid sequence encoding same).
This is to be used as is well understood in the art to locate the residue of interest. This is not always a strict counting exercise—attention must be paid to the context. For example, if the protein of interest is of a slightly different length, then location of the correct residue in that sequence corresponding to (for example) E664 may require the sequences to be aligned and the equivalent or corresponding residue picked, rather than simply taking the 664th residue of the sequence of interest. This is well within the ambit of the skilled reader.
Mutating may refer to the substitution or truncation or deletion of the residue, motif or domain referred to. Preferably, mutation means substitution. Thus, unless otherwise indicated expressly or by context, “mutation” may be taken to refer to substitution of the amino acid referred to herein.
Mutation may be effected at the polypeptide level, e.g., by synthesis of a polypeptide having the mutated sequence, or may be effected at the nucleotide level, e.g., by making a nucleic acid encoding the mutated sequence, which nucleic acid may be subsequently translated to produce the mutated polypeptide. Where no amino acid is specified as the replacement amino acid for a given mutation site, as a default alanine (A) may be used. Suitably the mutations used at particular site(s) are as set out herein.
A fragment is suitably at least 10 amino acids in length, suitably at least 25 amino acids, suitably at least 50 amino acids, suitably at least 100 amino acids, or suitably the majority of the polymerase polypeptide of interest, i.e., 387 amino acids or more, suitably at least 500 amino acids, suitably at least 600 amino acids, suitably at least 700 amino acids, suitably the entire 773 amino acids of the TgoT polB sequence.
Sequence VariationThe polymerase of the invention may comprise sequence changes relative to the wild type sequence in addition to the key mutations described in more detail herein. Specifically, the polymerase of the invention may comprise sequence changes at sites which do not significantly compromise the function or operation of the polymerase as described herein.
Polymerase function may be easily tested by operating the polymerase as described, such as in the examples section, in order to verify that function has not been abrogated or significantly altered.
Thus, provided that the polymerase retains its function which can be easily tested as set out herein, sequence variations may be made in the polymerase molecule relative to the wild type reference sequence.
Conservative substitutions may be made, for example according to Table 2, below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
In considering what mutations, substitutions, or other such changes might be made relative to the wild type sequence, retention of the function of the polymerase is paramount. Typically, conservative amino acid substitutions would be less likely to adversely affect the function. Suitably, the polymerase of the invention varies from the wild type sequence only by conservative amino acid substitutions except as discussed.
Sequence Homology/IdentityAlthough sequence homology can also be considered in terms of functional similarity (i.e., amino acid residues having similar chemical properties/functions), in the context of the present document it is preferred to express homology in terms of sequence identity.
Sequence comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate percent homology (such as percent identity) between two or more sequences.
Percent identity may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
Although this is a very simple and consistent method, it fails to take into consideration that, for example in an otherwise identical pair of sequences, one insertion or deletion will cause the subsequent amino acid residues to be out of alignment, thus potentially resulting in a large reduction in percent homology (percent identity) when a global alignment (an alignment across the whole sequence) is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology (identity) score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology/identity.
These more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. It is preferred, however, to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is −12 for a gap and −4 for each extension.
Calculation of maximum percent homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Univ. Wisconsin, US; Devereux et al., Nucl. Acids Res. 12:387(1984)). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and the GENEWORKS suite of comparison tools.
Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied. It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
Suitably, identity is assessed at the amino acid level over at least 400 or 500, preferably 600, 700, or even more amino acids with the relevant polypeptide sequence(s) disclosed herein, most suitably with the full length progenitor “true wild type” TgoT polB sequence of SEQ ID NO:1.
Suitably, homology should be considered with respect to one or more of those regions of the sequence known to be essential for protein function rather than non-essential neighbouring sequences. This is especially important when considering homologous sequences from distantly related organisms.
When considering conserved regions, suitably the 36% of residues common to both SEQ ID NO:1 and to the pol delta member of the polB enzyme family should be taken to be potentially important residues which are suitably not mutated in the polypeptide of the invention unless otherwise discussed. Thus, suitably the polypeptide of the invention has at least 36% identity to SEQ ID NO:1 and suitably the amino acid residues making up said at least 36% identity comprise the amino acid residues corresponding to those which are identical between SEQ IN NO:1 and the pol delta member of the polB enzyme family. Suitably, the polypeptide of the invention has at least 36% identity to SEQ ID NO:1 and has at least 36% identity to the pol delta member of the polB enzyme family.
The same considerations apply to nucleic acid nucleotide sequences.
Polynucleotides of the InventionPolynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus, in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli.
Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e., the vector is an expression vector. The term “operably linked” means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
Vectors of the invention may be transformed or transfected into a suitable host cell as described to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.
The vectors may be, for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. Vectors may be used, for example, to transfect or transform a host cell.
Control sequences operably linked to sequences encoding the protein of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
Protein Expression and PurificationProteins of the invention are typically made by recombinant means, for example as described below and in the Examples. Proteins may also be made by synthetic means, however, using techniques well known to skilled persons such as solid phase synthesis. Proteins of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Clearly, the fusion protein selected must not hinder the function of the polymerase of the invention.
Suitably, the polymerase of the invention is not fused to any sequence for purification since the polymerase of the invention may be advantageously purified based on its thermostable properties and/or using simple well known purification schemes as noted herein.
Host cells comprising polynucleotides of the invention may be used to express proteins of the invention. Host cells may be cultured under suitable conditions which allow expression of the proteins of the invention. Expression of the proteins of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG. Proteins of the invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.
Polymerase MutantsWe have identified a novel region of the polymerase enzyme which may be varied in order to provide orthogonal polymerase functions. Referring to the conventional structural model of the polymerase enzyme, the patch which has been identified is located in the “thumb” part of the enzyme. In the accepted model, the polymerase is thought of as a right “hand” extending around a DNA “rod” which passes through a central space defined by the structure of the enzyme. The region of the enzyme which we teach may be varied in order to provide the orthogonal polymerase functions described is the “thumb” part of the enzyme. In more detail, it is that part of the thumb at the exit point where the DNA “rod” leaves the enzymatic “hand”.
The region of the enzyme which we teach should be varied is the “thumb” region. The thumb region represents a single three-dimensional part of the enzyme. This single three-dimensional thumb structure is represented, however, by separate linear parts of the amino acid sequence. Within these linear parts, two patches are identified that specify orthogonal polymerase function. These two patches are designated patch 10A and patch 12 and are discussed in more detail below.
Patch 10APatch 10A corresponds to amino acids 651 to 679 of SEQ ID NO:1. Mutations in this patch can provide orthogonal polymerase activity for at least RNA, CeNA, or HNA. Patch 10A is considered the most important patch described herein.
Within patch 10A is a motif of special importance, which is the small beta-sheet region from amino acid residues (“aa”) 662 to 666. This region occupies a space near the nucleic acid backbone and is especially suitably mutated for polymerase(s) acting to produce RNA. In particular, residue E664 is especially suitable to mutate, for example E664Q or E664K; most preferred is E664K. This mutant is especially suitable for RNA production.
Although the 662-666 region (and E664 in particular) is especially important for RNA polymerase, the effect is not confined to RNA polymerase. For example, the E664 position is mutated in both C7 (SEQ ID NO:5; excellent CeNA pol activity) and 6G12 (SEQ ID NO:12; excellent HNA pol activity). Thus suitably, the orthogonal polymerase of the invention comprises an E664 mutation such as E664Q.
For RNA polymerase suitably a Y409 mutation is included such as Y409N, which is located outside the patch 10A. This has the advantage of increasing RNA pol activity.
Patch 12Patch 12 comprises amino acids 734 to 765 of SEQ ID NO:1. Mutation of patch 12 can provide orthogonal polymerase activity to at least HNA or CeNA. Patch 12 is important, but may be considered less important than Patch 10A.
Suitably, mutations may be made to one or other or both Patches 12 and 10A. If only one patch is to be mutated, suitably that is patch 10A.
Without wishing to be bound by theory, it is thought that the best mutations in these patches are those which alter the “thumb” structure of this region so that it no longer performs its gate keeping function at the exit of the nucleotide polymer from the enzyme. It is thought that by making these types of mutation, the natural “filtering” function of the enzyme is altered or disabled, thereby advantageously making the enzyme more promiscuous and more capable of producing orthogonal nucleic acid polymers.
Further polymerase mutations are found in the specific exemplary sequences provided but which lie outside of the key thumb domain patches 10A and 12. These may be defined by comparing exemplary sequence(s) for the polymerase(s) of the invention given in the sequence listing with the preferred backbone polypeptide given in SEQ ID NO:2, and by ignoring the mutations occurring in patch 10A (aa 651 to 679) and patch 12 (aa 734-765). The resulting mutations, which are identified by this process are each possible further optimising mutations which may be included in the polymerase(s) of the invention.
Important to HNA synthesis by the exemplary Pol6G12 are mutations in a flexible region of the polymerase thumb subdomain (Tgo: G586-T773), typically not visible in many PolB apo structures (e.g., 2JGU, 1Q8I, 1QHT, 1WNS), but resolved in the E. coli Pol II ternary complex (3MAQ). See Kim et al., Int. J. Biol. Macromol. 42:356 (2008); Rodriguez et al., J. Mol. Biol. 299:447 (2000); Hashimoto et al., J. Mol. Biol. 306:469 (2001); Wang & Yang, Cell 139:1279 (2009). Structurally, this region transverses the thumb along the face proximal to the exonuclease domain and makes close contacts with the nascent strand from +3 to +7. We posit that this structural region serves as an extension checkpoint and has been remodeled by mutation in Pol 6G12 to allow processive HNA synthesis. Indeed, other mutations in the same motif enable processive synthesis of other non-cognate nucleic acid polymers, pointing to a general specificity function for this polymerase region. Several of Pol6G12's other mutations (V589A, I610M, E664Q, Q665P, R668K, T676R, and A681S), cluster around the internal surface of the polymerase in proximity to the nascent strand. These mutations are likely to further reshape the duplex binding “funnel” of the polymerase to allow accommodation of the non-canonical HNA*DNA hybrid.
Reverse Transcriptase MutantsThe polymerase of the invention maybe mutated in order to endow it with reverse transcriptase activity. In other words, the polymerase of the invention may be mutated such as to allow the manufacture of a DNA polymer from an orthogonal nucleic acid template.
Suitably, in order to produce orthogonal reverse transcriptase activity the polymerase of the invention is mutated at I521 and/or Y388.
A shorter adaptive path led us to an HNA-RT. Although L408, the residue previously identified to be associated with RNA-RT activity in the related polB family polymerase Pfu, did not score highly; SCA identified four proximal residues with clear involvement in covariation networks, mutation of just one of which (I521L) yielded a polymerase with significant HNA reverse transcriptase activity. Surprisingly, I521 is not located in proximity to the HNA template strand, but rather packs onto residues 540-542 (DTD motif) in the conserved C-motif in the polymerase active site, proximal to the primer strand 3′ terminus. The I521L mutation may therefore not directly alter interactions of the polymerase with the HNA template strand but rather promote RT activity on a HNA template by repositioning of the nascent strand 3′ end to allow productive extension.
It is a specific advantage of the mutations described above that they permit the mutated polymerase to be used in the production of a DNA nucleic acid from an RNA template with the proof reading function of the polymerase preserved. In other words, the reverse transcriptase mutants of the invention may be used with RNA as the template whilst the exonuclease of the polymerase is intact (e.g., leaving at least D141 and E143 as functional residues such as wild type residues. This provides excellent results, for example one or even two orders of magnitude greater fidelity than conventional reverse transcriptase enzymes.
It should be noted that optimum activity on other orthogonal nucleic acid templates such as HNA or CeNA may require the proof reading/exonuclease function of the polymerase to be inhibited or ablated, for example by further mutation. Specifically, when the template is HNA then the proof reading/exonuclease function of the polymerase should be in activated.
The reverse transcriptase activity of the enzyme may be further optimised. For example, there are a number of known mutations which may be made in combination with the mutations of the invention in order to enhance activity when RNA is the template. For example, the asparagine mutation of the preferred D4N3 polymerase may be made in order to enhance the activity when used on RNA as the template.
Backbone MutationsWhether the enzyme of the invention is a non-DNA polymerase or a reverse transcriptase, there are a number of mutations that may advantageously be made to the backbone polypeptide.
One such mutation is the “therminator” mutant (New England Bio Labs) at position A485 of SEQ ID NO:1. Suitably, the backbone has an A485 mutation such as A485L. This has the advantage of enhancing incorporation of unnatural substrates.
Another such mutation is at position V93 of SEQ ID NO:1. Suitably, the backbone has a V93 mutation such as V93Q. This has the advantage of disabling read-ahead stalling which can occur, for example, when the template comprises uracil.
Another such mutation is at positions D141 and E143 of SEQ ID NO:1. Suitably, the backbone has a D141 mutation such as D141A; suitably, the backbone has an E143 mutation, such as, E143A; most suitably, the backbone has both D141 and E143 mutations, such as, D141A and E143A. This has the advantage of disabling the exonuclease function of the enzyme. This further enhances incorporation of unnatural substrates.
The mutations mentioned are mutually compatible; in other words the polypeptide of the invention may have each of the backbone mutations in the same polypeptide. An example of this is in SEQ ID NO:2. This is sometimes referred to as the “wild type” sequence and may be regarded as an excellent example of a starting polymerase backbone into which the mutations of the invention may be introduced. Thus, suitably all four of the preferred backbone mutations V93Q, A485L, D141A, and E143A are present in the polypeptides of the invention.
In a strict sense of course it will be noted that the sequence of SEQ ID NO:2 is not the true “wild type” because it has these four mutations in the backbone already. For ease of reference, the true wild type sequence is given as SEQ ID NO:1; this sequence is referred to as the “true wild type” herein for clarity.
Other conventional mutations may be applied to the polymerase/reverse transcriptase. Moreover, other optimising mutations may be made as appropriate.
TruncationsTruncations of the overall full-length polymerase enzyme of the invention may be made if desired. Suitably, full length polymerase polypeptide is used as the backbone polypeptide, such as, full length TgoT polymerase 1-773 as shown in the attached sequence listing. Any truncations used should be carefully checked for activity. This may be easily done by assaying the enzyme(s) as described herein.
PurificationPolymerases of the invention are advantageously thermo-stable. By expressing these polymerases in a conventional (non thermo-stable) host strain, purification is advantageously simplified. For example, when the polymerases of the invention are expressed in a conventional non thermo-stable host cell, approximately 90% purity may be obtained simply by heating the host cells to 99° C. followed by centrifugal removal of cellular debris. Higher purity levels may easily be obtained for example by subjecting the heat treated soluble fraction of the host cell to ion exchange and/or heparin column purifications.
Suitably, the polymerase of the invention is not fused to any other polypeptide.
Suitably, the polymerase of the invention is not tagged with any further polypeptides or fusions.
It is an advantage of the invention that mutations directed to the particular patches discussed may be substitutions to any of a wide range of amino acids without loss of function of the polymerase. The patches defined are extremely tolerant of amino acid changes.
FidelityIt is clearly important that sufficient fidelity is maintained for accurate production (or reproduction) of the orthogonal nucleic acid polymers. Suitably, polymerases of the invention retain at least 95% fidelity. Fidelity (error threshold) may be taken as the number of errors introduced divided by the number of nucleotides polymerised. In other words, an error rate of 1% equates to the introduction of one error for every 100 nucleotides polymerised. In fact, the polymerases of the invention attain a much better fidelity than this. An error rate of 5% or less is considered as the minimum useful fidelity level for the polymerases of the invention; suitably the polymerases of the invention have an error rate of 4% or less; suitably 3% or less; suitably 2% or less; suitably 1% or less.
Unless otherwise stated, fidelity may be assessed as aggregate fidelity (e.g., DNA-3NA-DNA) which thus encompasses two conversion events (DNA-3NA and 3NA-DNA); the figures should be adjusted or interpreted accordingly.
Compartmentalised Self-Replication TechnologyThe techniques of directed evolution and compartmentalized self-replication are detailed in GB 97143002 and GB 98063936 and GB 01275643. These documents are herein incorporated by reference.
The inventors modified the methods of compartmentalised self-tagging and surprisingly generated DNA polymerases which exhibited an expanded substrate range as herein defined.
Further details of the method of compartmentalised self-tagging in general are given below. Of particular importance in the selection of polymerases which exhibit an enhanced ability to synthesize orthogonal nucleic acid polymers such as exemplified for HNA or CeNA herein (as compared with the polymerase from which they are derived) is that the compartmentalised self-tagging method was modified. These modifications are detailed below.
(i) Microcapsules
The microcapsules used in some applications of the invention require appropriate physical properties to allow the working of the invention.
First, to ensure that the nucleic acids and gene products may not diffuse between microcapsules, the contents of each microcapsule must be isolated from the contents of the surrounding microcapsules, so that there is no or little exchange of the nucleic acids and gene products between the microcapsules over the timescale of the experiment.
Second, microcapsule methods of the present invention require that there are only a limited number of nucleic acids per microcapsule. This ensures that the gene product of an individual nucleic acid will be isolated from other nucleic acids. Thus, coupling between nucleic acid and gene product will be highly specific. The enrichment factor is greatest with, on average, one or fewer nucleic acids per microcapsule, the linkage between nucleic acid and the activity of the encoded gene product being as tight as is possible, since the gene product of an individual nucleic acid will be isolated from the products of all other nucleic acids. Even if the theoretically optimal situation of, on average, a single nucleic acid or less per microcapsule is not used, a ratio of 5, 10, 50, 100 or 1000 or more nucleic acids per microcapsule may prove beneficial in sorting a large library. Subsequent rounds of sorting, including renewed encapsulation with differing nucleic acid distribution, will permit more stringent sorting of the nucleic acids. Preferably, there is a single nucleic acid, or fewer, per microcapsule.
Third, the formation and the composition of the microcapsules must not abolish the function of the machinery the expression of the nucleic acids and the activity of the gene products.
Consequently, any microencapsulation system used must fulfil these three requirements. The appropriate system(s) may vary depending on the precise nature of the requirements in each application of the invention, as will be apparent to the skilled person.
A wide variety of microencapsulation procedures are available (see Benita, 1996) and may be used to create the microcapsules used in accordance with the present invention. Indeed, more than 200 microencapsulation methods have been identified in the literature (Finch, 1993).
These include membrane enveloped aqueous vesicles such as lipid vesicles (liposomes) (New, 1990) and non-ionic surfactant vesicles (van Hal et al., 1996). These are closed-membranous capsules of single or multiple bilayers of non-covalently assembled molecules, with each bilayer separated from its neighbour by an aqueous compartment. In the case of liposomes the membrane is composed of lipid molecules; these are usually phospholipids but sterols such as cholesterol may also be incorporated into the membranes (New, 1990). A variety of enzyme-catalysed biochemical reactions, including RNA and DNA polymerisation, can be performed within liposomes (Chakrabarti et al., 1994; Oberholzer et al., 1995a; Oberholzer et al., 1995b; Walde et al., 1994; Wick & Luisi, 1996).
With a membrane-enveloped vesicle system much of the aqueous phase is outside the vesicles and is therefore non-compartmentalised. This continuous, aqueous phase should be removed or the biological systems in it inhibited or destroyed (for example, by digestion of nucleic acids with DNase or RNase) in order that the reactions are limited to the microcapsules (Luisi et al., 1987).
Enzyme-catalysed biochemical reactions have also been demonstrated in microcapsules generated by a variety of other methods. Many enzymes are active in reverse micellar solutions (Bru & Walde, 1991; Bru & Walde, 1993; Creagh et al., 1993; Haber et al., 1993; Kumar et al., 1989; Luisi & B., 1987; Mao & Walde, 1991; Mao et al., 1992; Perez et al., 1992; Walde et al., 1994; Walde et al., 1993; Walde et al., 1988) such as the AOT-isooctane-water system (Menger & Yamada, 1979).
Microcapsules can also be generated by interfacial polymerisation and interfacial complexation. Whateley, 1996. Microcapsules of this sort can have rigid, nonpermeable membranes, or semipermeable membranes. Semipermeable microcapsules bordered by cellulose nitrate membranes, polyamide membranes and lipid-polyamide membranes can all support biochemical reactions, including multienzyme systems. Chang, 1987; Chang, 1992; Lim, 1984. Alginate/polylysine microcapsules (Lim & Sun, 1980), which can be formed under very mild conditions, have also proven to be very biocompatible, providing, for example, an effective method of encapsulating living cells and tissues (Chang, 1992; Sun et al., 1992).
Non-membranous microencapsulation systems based on phase partitioning of an aqueous environment in a colloidal system, such as an emulsion, may also be used.
Preferably, the microcapsules of the present invention are formed from emulsions; heterogeneous systems of two immiscible liquid phases with one of the phases dispersed in the other as droplets of microscopic or colloidal size. Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant, 1984.
(ii) Emulsions
Emulsions may be produced from any suitable combination of immiscible liquids. Preferably the emulsion of the present invention has water (containing the biochemical components) as the phase present in the form of finely divided droplets (the disperse, internal or discontinuous phase) and a hydrophobic, immiscible liquid (an “oil”) as the matrix in which these droplets are suspended (the nondisperse, continuous or external phase). Such emulsions are termed “water-in-oil” (W/O). This has the advantage that the entire aqueous phase containing the biochemical components is compartmentalised in discreet droplets (the internal phase). The external phase, being a hydrophobic oil, generally contains none of the biochemical components and hence is inert.
The emulsion may be stabilised by addition of one or more surface-active agents (surfactants). These surfactants are termed emulsifying agents and act at the water/oil interface to prevent (or at least delay) separation of the phases. Many oils and many emulsifiers can be used for the generation of water-in-oil emulsions; a recent compilation listed over 16,000 surfactants, many of which are used as emulsifying agents (Ash & Ash, 1993). Suitable oils include light white mineral oil and non-ionic surfactants (Schick, 1966), such as sorbitan monooleate (Span™80; ICI) and polyoxyethylenesorbitan monooleate (Tween™ 80; ICI) and Triton-X-100.
The use of anionic surfactants may also be beneficial. Suitable surfactants include sodium cholate and sodium taurocholate. Particularly preferred is sodium deoxycholate, preferably at a concentration of 0.5% w/v, or below. Inclusion of such surfactants can in some cases increase the expression of the nucleic acids and/or the activity of the gene products. Addition of some anionic surfactants to a non-emulsified reaction mixture completely abolishes translation. During emulsification, however, the surfactant is transferred from the aqueous phase into the interface and activity is restored. Addition of an anionic surfactant to the mixtures to be emulsified ensures that reactions proceed only after compartmentalisation.
Creation of an emulsion generally requires the application of mechanical energy to force the phases together. There are a variety of ways of doing this which utilise a variety of mechanical devices, including stirrers (such as magnetic stir-bars, propeller and turbine stirrers, paddle devices and whisks), homogenisers (including rotor-stator homogenisers, high-pressure valve homogenisers and jet homogenisers), colloid mills, ultrasound and “membrane emulsification” devices. Becher, 1957; Dickinson, 1994.
Aqueous microcapsules formed in water-in-oil emulsions are generally stable with little if any exchange of nucleic acids or gene products between microcapsules. Additionally, we have demonstrated that several biochemical reactions proceed in emulsion microcapsules. Moreover, complicated biochemical processes, notably gene transcription and translation are also active in emulsion microcapsules. The technology exists to create emulsions with volumes all the way up to industrial scales of thousands of litres. Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant, 1984.
The preferred microcapsule size will vary depending upon the precise requirements of any individual selection process that is to be performed according to the present invention. In all cases, there will be an optimal balance between gene library size, the required enrichment and the required concentration of components in the individual microcapsules to achieve efficient expression and reactivity of the gene products.
Details of emulsion/s used when performing the method of the present invention are provided in the Examples.
(iii) Expression within Microcapsules
The processes of expression usually occur within each individual microcapsule provided by the present invention. Both in vitro transcription and coupled transcription-translation become less efficient at sub-nanomolar DNA concentrations. Because of the requirement for only a limited number of DNA molecules to be present in each microcapsule, this therefore sets a practical upper limit on the possible microcapsule size. Preferably, the mean volume of the microcapsules is less than 5.2×10−16 m3 (corresponding to a spherical microcapsule of diameter less than 10 μm), more preferably less than 6.5×10−17 m3 (5 μm), more preferably about 4.2×10−18 m3 (2 μm) and ideally about 9×10−18 m3 (2.6 μm).
The effective DNA or RNA concentration in the microcapsules may be artificially increased by various methods that will be well-known to those versed in the art. These include, for example, the addition of volume excluding chemicals such as polyethylene glycols (PEG) and a variety of gene amplification techniques, including transcription using RNA polymerases including those from bacteria such as E. coli (Roberts, 1969; Blattner & Dahlberg, 1972; Roberts et al., 1975; Rosenberg et al., 1975), eukaryotes (Weil et al., 1979; Manley et al., 1983), and bacteriophage such as T7, T3 and SP6 (Melton et al., 1984); the polymerase chain reaction (PCR) (Saiki et al., 1988); Q3 replicase amplification (Miele et al., 1983; Cahill et al., 1991; Chetverin & Spirin, 1995; Katanaev et al., 1995); the ligase chain reaction (LCR) (Landegren et al., 1988; Barany, 1991); and self-sustained sequence replication system (Fahy et al., 1991); and strand displacement amplification (Walker et al., 1992). Even gene amplification techniques requiring thermal cycling such as PCR and LCR could be used if the emulsions and the in vitro transcription or coupled transcription-translation systems are thermostable (for example, the coupled transcription-translation systems could be made from a thermostable organism such as Thermus aquaticus).
Increasing the effective local nucleic acid concentration enables larger microcapsules to be used effectively. This allows a preferred practical upper limit to the microcapsule volume of about 5.2×10−16 m3 (corresponding to a sphere of diameter 10 μm).
The microcapsule size must be sufficiently large to accommodate all of the required components of the biochemical reactions that are needed to occur within the microcapsule. For example, in vitro, both transcription reactions and coupled transcription-translation reactions require a total nucleoside triphosphate concentration of about 2 mM.
For example, in order to transcribe a gene to a single short RNA molecule of 500 bases in length, this would require a minimum of 500 molecules of nucleoside triphosphate per microcapsule (8.33×10−22 moles). In order to constitute a 2 mM solution, this number of molecules must be contained within a microcapsule of volume 4.17×10−19 litres (4.17×10−22 m3 which if spherical would have a diameter of 93 nm.
Furthermore, particularly in the case of reactions involving translation, it is to be noted that the ribosomes necessary for the translation to occur are themselves approximately 20 nm in diameter. Hence, the preferred lower limit for microcapsules is a diameter of approximately 100 nm.
Therefore, the microcapsule volume is preferably of the order of between 5.2×10−22 m3 and 5.2×10−16 m3 corresponding to a sphere of diameter between 0.1 μm and 10 μm, more preferably of between about 5.2×10−19 m3 and 6.5×10−17 m3 1 μm and 5 μm). Sphere diameters of about 2.6 μm are most advantageous.
It is no coincidence that the preferred dimensions of the compartments (droplets of 2.6 μm mean diameter) closely resemble those of bacteria, for example, Escherichia are 1.1-1.5×2.0-6.0 μm rods and Azotobacter are 1.5-2.0 μm diameter ovoid cells. In its simplest form, Darwinian evolution is based on a “one genotype one phenotype” mechanism. The concentration of a single compartmentalised gene, or genome, drops from 0.4 nM in a compartment of 2 μm diameter, to 25 pM in a compartment of 5 μm diameter. The prokaryotic transcription/translation machinery has evolved to operate in compartments of ˜1-2 μm diameter, where single genes are at approximately nanomolar concentrations. A single gene, in a compartment of 2.6 μm diameter is at a concentration of 0.2 nM. This gene concentration is high enough for efficient translation. Compartmentalisation in such a volume also ensures that even if only a single molecule of the gene product is formed it is present at about 0.2 nM, which is important if the gene product is to have a modifying activity of the nucleic acid itself. The volume of the microcapsule should thus be selected bearing in mind not only the requirements for transcription and translation of the nucleic acid/nucleic acid, but also the modifying activity required of the gene product in the method of the invention.
The size of emulsion microcapsules may be varied simply by tailoring the emulsion conditions used to form the emulsion according to requirements of the selection system. The larger the microcapsule size, the larger is the volume that will be required to encapsulate a given nucleic acid/nucleic acid library, since the ultimately limiting factor will be the size of the microcapsule and thus the number of microcapsules possible per unit volume.
The size of the microcapsules is selected not only having regard to the requirements of the transcription/translation system, but also those of the selection system employed for the nucleic acid/nucleic acid construct. Thus, the components of the selection system, such as a chemical modification system, may require reaction volumes and/or reagent concentrations which are not optimal for transcription/translation. As set forth herein, such requirements may be accommodated by a secondary re-encapsulation step; moreover, they may be accommodated by selecting the microcapsule size in order to maximise transcription/translation and selection as a whole. Empirical determination of optimal microcapsule volume and reagent concentration, for example as set forth herein, is preferred.
A “nucleic acid” in accordance with the present invention is, preferably, a molecule or construct selected from the group consisting of a DNA molecule, an RNA molecule, a partially or wholly artificial nucleic acid molecule consisting of exclusively synthetic or a mixture of naturally occurring and synthetic bases. Any one of the foregoing may be linked to a polypeptide.
The nucleic acid portion of the nucleic acid may comprise suitable regulatory sequences, such as those required for efficient expression of the gene product, for example promoters, enhancers, translational initiation sequences, polyadenylation sequences, splice sites and the like.
(iv) Product Selection
Details of a preferred method of performing the method of the invention are provided in the Examples. However, those skilled in the art will appreciate that the examples given are non-limiting and methods for product selection are discussed in more general terms below.
A ligand or substrate can be connected to the nucleic acid by a variety of means that will be apparent to those skilled in the art (see, e.g., Hermanson, 1996). According to the method of the present invention, the ligand or substrate is a ‘detection agent label’, preferably a dye-labelled nucleotide analogue, in particular Cy3CTP and/or Cy5CTP.
Sorting can be by any method which allows the preferential separation, amplification or survival of the detection agent labelled nucleic acid. Examples include selection by binding (including techniques based on magnetic separation, for example using Dynabeads™), and by resistance to degradation (for example by nucleases, including restriction endonucleases).
When all reactions are stopped and the microcapsules are combined, the nucleic acids encoding the active engineered polymerases selected can be enriched using an antibody or other molecule which binds, or reacts specifically with the “detection agent label”. Although both substrates and product have the detection agent label, only the nucleic acids encoding active gene product will co-purify.
The terms “isolating”, “sorting” and “selecting”, as well as variations thereof, are used herein. Isolation, according to the present invention, refers to the process of separating an entity from a heterogeneous population, for example a mixture, such that it is substantially, preferably totally, free of at least one substance with which it was associated before the isolation process. In a preferred embodiment, isolation refers to purification of an entity essentially to homogeneity. Sorting of an entity refers to the process of preferentially isolating desired entities over undesired entities. In as far as this relates to isolation of the desired entities, the terms “isolating” and “sorting” are equivalent. The method of the present invention permits the sorting of desired nucleic acids from pools (libraries or repertoires) of nucleic acids which contain the desired nucleic acid. Selecting is used to refer to the process (including the sorting process) of isolating an entity according to a particular property thereof.
Initial selection of a nucleic acid from a nucleic acid library (for example a mutant Pfu library) using the present invention will in most cases require the screening of a large number of variant nucleic acids. Libraries of nucleic acids can be created in a variety of different ways, including the following.
Pools of naturally occurring nucleic acids can be cloned from genomic DNA or cDNA (Sambrook et al., 1989); for example, mutant Tgo libraries or other DNA polymerase libraries, made by PCR amplification repertoires of Tgo or other DNA polymerase genes have proved very effective sources of DNA polymerase fragments. Further details are given in the Examples.
Libraries of genes can also be made by encoding all (see, e.g., Smith, 1985; Parmley & Smith, 1988) or part of genes (see, e.g., Lowman et al., 1991) or pools of genes (see, e.g., Nissim et al., 1994) by a randomised or doped synthetic oligonucleotide. Libraries can also be made by introducing mutations into a nucleic acid or pool of nucleic acids “randomly” by a variety of techniques in vivo, including; using “mutator strains” of bacteria such as E. coli mutD5. Liao et al., 1986; Yamagishi et al., 1990; Low et al., 1996. Random mutations can also be introduced both in vivo and in vitro by chemical mutagens, and ionising or UV irradiation (see Friedberg et al., 1995), or incorporation of mutagenic base analogues (Freese, 1959; Zaccolo et al., 1996). “Random” mutations can also be introduced into genes in vitro during polymerization, for example, by using error-prone polymerases. Leung et al., 1989. Further diversification can be introduced by using homologous recombination either in vivo (see Kowalczykowski et al., 1994) or in vitro (Stemmer, 1994a; Stemmer, 1994b).
(v) Microcapsules/Sorting
In addition to the nucleic acids described above, the microcapsules according to the invention will comprise further components required for the sorting process to take place. Other components of the system will for example comprise those necessary for transcription and/or translation of the nucleic acid. These are selected for the requirements of a specific system from the following; a suitable buffer, an in vitro transcription/replication system and/or an in vitro translation system containing all the necessary ingredients, enzymes and cofactors, RNA polymerase, nucleotides, nucleic acids (natural or synthetic), transfer RNAs, ribosomes and amino acids, and the substrates of the reaction of interest in order to allow selection of the modified gene product.
A suitable buffer will be one in which all of the desired components of the biological system are active and will therefore depend upon the requirements of each specific reaction system. Buffers suitable for biological and/or chemical reactions are known in the art and recipes provided in various laboratory texts, such as Sambrook et al., 1989.
The in vitro translation system will usually comprise a cell extract, typically from bacteria (Zubay, 1973; Zubay, 1980; Lesley et al., 1991; Lesley, 1995), rabbit reticulocytes (Pelham &_Jackson, 1976), or wheat germ (Anderson et al., 1983). Many suitable systems are commercially available (for example from Promega) including some which will allow coupled transcription/translation (all the bacterial systems and the reticulocyte and wheat germ TNT™ extract systems from Promega). The mixture of amino acids used may include synthetic amino acids if desired, to increase the possible number or variety of proteins produced in the library. This can be accomplished by charging tRNAs with artificial amino acids and using these tRNAs for the in vitro translation of the proteins to be selected. Ellman et al., 1991; Benner, 1994; Mendel et al., 1995.
After each round of selection the enrichment of the pool of nucleic acids for those encoding the molecules of interest can be assayed by non-compartmentalised in vitro transcription/replication or coupled transcription-translation reactions. The selected pool is cloned into a suitable plasmid vector and RNA or recombinant protein is produced from the individual clones for further purification and assay.
(vi) Microcapsule Identification
Microcapsules may be identified by virtue of a change induced by the desired gene product which either occurs or manifests itself at the surface of the microcapsule or is detectable from the outside as described in section (v) (Microcapsule sorting). This change, when identified, is used to trigger the modification of the gene within the compartment. In a preferred aspect of the invention, microcapsule identification relies on a change in the optical properties of the microcapsule resulting from a reaction leading to luminescence, phosphorescence or fluorescence within the microcapsule. Modification of the gene within the microcapsules would be triggered by identification of luminescence, phosphorescence or fluorescence. For example, identification of luminescence, phosphorescence or fluorescence can trigger bombardment of the compartment with photons (or other particles or waves) which leads to modification of the nucleic acid. A similar procedure has been described previously for the rapid sorting of cells (Keij et al., 1994). Modification of the nucleic acid may result, for example, from coupling a molecular “fluorescent detection agent label,” caged by a photolabile protecting group to the nucleic acids: bombardment with photons of an appropriate wavelength leads to the removal of the cage. Afterwards, all microcapsules are combined and the nucleic acids pooled together in one environment. Nucleic acids encoding gene products exhibiting the desired activity can be selected by affinity purification using a molecule that specifically binds to, or reacts specifically with, the “fluorescent label.”
(vii) Multi-Step Procedure
It will also be appreciated that according to the present invention, it is not necessary for all the processes of transcription/replication and/or translation, and selection to proceed in one single step, with all reactions taking place in one microcapsule. The selection procedure may comprise two or more steps. First, transcription/replication and/or translation of each nucleic acid of a nucleic acid library may take place in a first microcapsule. Each gene product is then linked to the nucleic acid which encoded it (which resides in the same microcapsule). The microcapsules are then broken, and the nucleic acids attached to their respective gene products optionally purified. Alternatively, nucleic acids can be attached to their respective gene products using methods which do not rely on encapsulation; for example, phage display (Smith, G. P., 1985), polysome display (Mattheakkis et al., 1994), RNA-peptide fusion (Roberts & Szostak, 1997), or lac repressor peptide fusion (Cull, et al., 1992).
In the second step of the procedure, each purified nucleic acid attached to its gene product is put into a second microcapsule containing components of the reaction to be selected. This reaction is then initiated. After completion of the reactions, the microcapsules are again broken and the modified nucleic acids are selected. In the case of complicated multistep reactions in which many individual components and reaction steps are involved, one or more intervening steps may be performed between the initial step of creation and linking of gene product to nucleic acid, and the final step of generating the selectable change in the nucleic acid.
(viii) Amplification
In all the above configurations, genetic material comprised in the nucleic acids may be amplified and the process repeated in iterative steps. Amplification may be by the polymerase chain reaction (Saiki et al., 1988) or by using one of a variety of other gene amplification techniques including; Qβ replicase amplification (Cahill, Foster & Mahan, 1991; Chetverin & Spirin, 1995; Katanaev, Kurnasov & Spirin, 1995); the ligase chain reaction (LCR) (Landegren et al., 1988; Barany, 1991); the self-sustained sequence replication system (Fahy, Kwoh & Gingeras, 1991) and strand displacement amplification (Walker et al., 1992).
The invention is now described by way of example. These examples are intended to be illustrative, and are not intended to limit the appended claims.
EXAMPLES Example 1: Development of a Novel High-Throughput Screening System for XNA SynthesisAn effective scalable screen is an essential step to test individual clones and to select clones with improved activity from a population. The polymerase ELISA endpoint assay, developed by Ong and colleagues (Ong et al., J. Mol. Biol. 361:537 (2006)), is summarised in
The polymerase ELISA endpoint assay was used to screen A-motif TgoT mutants selected in the second round of CSR (both single nucleotide and dinucleotide substitution selections) with mixed results. A number of mutants were identified by the assay, which used a hairpin that required ten CeRTP incorporations prior to the first dUTP-DIG one. Further screening with primer extension reactions showed that few of the isolated mutants had comparable activity to wild-type TgoT and of the 40 isolated mutants, only three showed improvement in activity.
A possible explanation is that the labelled-nucleotide incorporation does not correlate as well to activity in PolB-type polymerases (e.g., TgoT) as it does to activity in PolA-type ones (e.g., Taq). The more accommodating catalytic site of PolB enzymes or the unnatural nucleotides being used may favour labelled-nucleotide incorporation leading to a higher signal in the ELISA that does not represent the desired polymerase activity.
One alternative further explored was to separate the extension from the detection steps. As described in
A number of steps (shown in
The results of a successful screen carried out with the two-step polymerase activity ELISA are shown in
A typical reaction is carried out as follows: An annealing master reaction containing biotinylated primer (and fluorescently labelled primer is direct detection is required), excess template (usually 4:1), nucleotides (optimum results above 100 μM final concentration) and polymerase buffer is made, heated and cooled to allow primer annealing. Remaining reaction components (enzyme, extra buffer or additional supplements) are added and reaction carried out (either as a single extension cycle or with thermocycling akin to PCR). Reaction time is adjusted according to the enzyme's level of activity and desired screening stringency. Each 10 μl reaction is transferred to a streptavidin-coated well (100 μl PBST+10 μl reaction+90 μl PBST) and incubated at room temperature for 30 min. The supernatant is discarded and each well washed thrice with PBST (Phosphate-buffered saline supplemented with 0.1% Tween20). The buffer is removed and the plate incubated in 100 mM NaOH for 30 sec. Wells are washed once with 100 μl mM NaOH and a second time with PBST. PBST is removed and 200 μl of probe solution (diluted in PBST to 0.2 μM final concentration) is added to each well and incubated at room temperature for 30 min. Wells are washed thrice with PBST and the antibody against the probe added. Reaction then proceeds as a standard ELISA.
Example 2: Development of a Selection System for XNA SynthesisDevelopment of a selection system suitable for isolating a CeNA or an HNA synthetase was carried out in parallel with selection itself. Thus, this section focuses on the development of the system and on the unsuccessful attempts for selecting a synthetase. The successful selection of the HNA synthetase will be described in the subsequent sections.
Initially, selection was attempted by short-patch compartmentalised self-replication (spCSR) (Ong et al., 2006); a technique previously developed in our lab. But this approach proved unproductive.
Consequently, a second selection methodology was developed: compartmentalised self-tagging (CST). As replication is decoupled from incorporation, CST is more likely to isolate successful synthetases due to the lowered selection barrier. In addition, the genetic information being selected remains as DNA throughout and is more likely to be successfully recovered.
The basic principles of CST have been described. Further optimisation of selection conditions and of the methodology itself led to the development of a new version of the CST strategy (CST2.0), summarized in
The key differences between the two variations lie on the primer of choice (random rather than specific, poor binder rather than stable binder) and on the downstream sample processing (formamide wash rather than 37° C. incubation). These changes in the methodology were key to the improvements observed.
A simple in silico model of the CST experiment suggested that amplification of total recovered DNA did not need to correlate with activity for libraries in which improved clones were rare. Once all modifications were incorporated, a model selection comparing wild-type and an inactive frame-shift mutant, yielded a 3-fold poorer enrichment than pre-modification (10-fold wt enrichment rather than the previous 30-fold observed), for instance. But that would then be expected if efficient selection required higher than wild-type activity levels.
A single round of selection on a 10A motif library (hNTP selection using an N6 primer) was carried out and a number of individual clones assessed for hNTP activity. The results, summarised in Table 3, confirmed that selection was indeed taking place and a number of improved polymerases were isolated, as shown in
Polymerase activity for each mutant was determined with the polymerase ELISA previously developed. In the case of TgoT (wild-type) under the reaction conditions used, ELISA signal was around 0.05 (hNTP) and 3.0 (dNTP). Polymerases with dNTP activity below 0.2 were deemed inactive. Polymerases with hNTP signal between 0.1 and 0.9 were deemed wild-type like. Enzymes above the 0.9 threshold were deemed improved. Improved enzymes were selected for downstream screening and analysis. The two populations were compared using a χ2 test (χ2=0.019, 2 d.f.)
Example 3: DNA Polymerase Library DesignInitially, three libraries in both TgoT and 9° NT polymerases were made targeted against the three conserved motifs that make the polymerase active site: A, B and C motifs. The libraries tried to encompass any phylogenetic diversity available in the region as well as low levels of mutations in all but the functionally important residues (e.g., catalytic aspartate). Similar libraries in Pfu had successfully been used to isolate a polymerase capable of incorporation and replication of Cy-labelled nucleotides. Ramsay et al., J. Am. Chem. Soc. 132:5096 (2010).
Although the three motifs line the active site of the polymerase and the incoming nucleotide pocket, it was clear that their optimisation could not be sufficient to obtain a processive polymerase. As a result, further libraries were designed using a similar approach to introduce mutations.
As only two polymerases of the PolB family have been crystallized with DNA to date (in a processive conformation)—Rb69 (Franklin et al., Cell 105:657 (2001)) and more recently phi29 Berman et al., EMBO J. 26:3494 (2007)) polymerases, shown in
Initial primer extension reactions identified that TgoT would efficiently incorporate a natural nucleotide after a cyclohexenyl nucleotide prior to stalling. Hence, further residues to be explored were identified by locating all residues within 10 Å of the +1 incorporation in the primer strand of the Rb69 polymerase structure. Water molecules identified in that search were then used to search a further 5 Å sphere. Residues identified on Rb69 structure were mapped to the available Tgo structure. Nine patches were identified in the Tgo sequence that would be expected to lie in the vicinity of the nascent helix. Two more patches were identified by structural comparison of the thumb structures of Rb69 and Tgo. The motifs identified were then mapped to phi29 to confirm their location; all but the thumb motifs (phi29 has a structurally divergent thumb) were successfully mapped.
The resultant eleven patches were used as the basis to design a further 18 libraries, shown in
Diversity was introduced through oligonucleotide synthesis of the primers used to generate the library. A typical amplification reaction (100 μl) would be carried out with Roche's Expand High Fidelity using the conditions recommended by the manufacturer and our wild-type plasmid DNA. The PCR would be carried out as a “touchdown” reaction (annealing temperature decreased after each extension cycle). As the primers have a small overlap downstream of BsaI sites, it allows the whole plasmid to be amplified and cloned seamlessly following BsaI (NEB) and DpnI (NEB) restriction reactions, and subsequent ligation with T4 DNA polymerase (NEB). All reactions were carried out according to the manufacturer's recommendations. For first round libraries, a further amplification step using phi29 (NEB) was carried out using N6 oligos as primers and reaction conditions as recommended by the manufacturer.
It is important to notice that the polymerase we have termed “wild-type”, and that makes the basis of all synthesized libraries, has a number of mutations present which are not present in the polymerase gene as isolated from Thermococcus gorgonarius. Our “wild-type” harbours four mutations: V93Q (introduced to reduce the polymerase read-ahead function that causes stalls if uracil is detected on the template) (Fogg et al., Nat. Str. Biol. 9:922 (2002)), D141A and E143A (introduced to inactivate the 3′→5′ exonuclease domain) (Joyce & Derbyshire, Meth. Enzymol. 262:3 (1995); Blanco et al., Gene 112:139 (2002); Bernad et al., Cell 59:219 (1989)), and A485L (commercially available as Therminator, but shown to improve incorporation of unnatural substrates in B-family polymerases) (Gardner & Jack, Nucl. Acids Res. 27:2545 (1999)).
Once synthesized by PCR, the ligated plasmids were transformed and a small sample of clones (between 10 and 30 clones per library) sequenced to confirm identity and estimate library diversity. Once gene integrity (dNTP incorporation activity and sequencing results) and library diversity were estimated to be high, large scale transformations were carried out to generate the libraries used for the remainder of the project.
Example 4: Selected Clones with Enhanced HNA/CeNA/RNA Polymerase ActivityCrude lysates of all available libraries normalised for their dNTP incorporation activity were used to identify which ones had the highest potential to house possible synthetases. Selection was carried out in those libraries.
Single round selections were carried out in motif libraries B−, 8, 9, 10A, 10B, 10C, 11 and 12 for both CeNTP and hNTP incorporations. Small-scale screens were carried out on motifs 8, 10A and 12. Although CeNTP incorporation improvement was detected, hNTP improvements were substantially higher. Those results, confirmed that CST was suitable for synthetase selection.
Mutants with improved incorporation parameters for both HNA and CeNA were isolated (see the Sequence Listing) validating the CST2.0 approach, and a further round of CST selection was carried out on the resulting 10A library. CST selections were also carried out on the 10A motif library (made in the context of a steric gate mutant Y409N) to isolate enzymes that could outperform the wild-type TgoT in synthesising RNA (see Examples 7-12).
Example 5: Design and Evolution of Polymerase for Processive Synthesis of CeNA and/or HNAAs improved mutants were isolated from different motif libraries, we investigated whether the mutations being identified could be additive. For that, we crossed two motif 12 mutants and four motif 10A ones to obtain the eight possible chimeras.
Although mutant activity always exceeded wild-type activity, no significant gain was attained by any of the chimeras. This suggests that mutations in motif 10A and motif 12 target a similar function in the polymerase hence 10A/12 chimeras do not lead to any gain in activity.
Another possibility investigated was the cross-substrate activity. Selection for CeNTP or hNTP incorporation may alter the enzyme's ability to recognise a modified nucleic acid structure, which may be similar for hNTP and CeNTP. While no gain in activity was observed for hNTP-selected mutants on CeNTP incorporation, the complementary experiment (hNTP incorporation by CeNTP-selected enzymes) identified a number of clones with even higher hNTP activity, as shown in
Similarly, the isolated mutants were also tested for rNTP (ribonucleotide) incorporation. Despite the presence of the Y409 steric gate (Gardner & Jack, 1999) in all the selected polymerases which greatly reduces rNTP incorporation, D4 showed considerable incorporation of rNTPs, as shown in
Sequence analysis of the 18 isolated motif 10A mutants have identified two clusters of mutations within the introduced diversity, targeting residues likely to be involved in maintaining local protein structure as well as residues likely to be in close proximity to the extending primer phosphate backbone (based on a structural alignment with RB69 polB) and thus of potential functional importance.
Example 6: A Processive HNA Polymerase: 6G12A second round of CST (CeNTP selection) was carried out on a 10A motif library (initially selected with CeNTP) to investigate the effect of further selection rounds on the library activity. One screen identified a mutant with improved CeNTP incorporation potential (marginally better than all the mutants identified in the first round) that also showed significant cross-substrate (hNTP) activity: 6G12.
A total of fourteen mutations have accumulated in 6G12, including six which lie outside of the selected motif. All mutations, when mapped to a RB69 structure, line the inner surface of the polymerase in close contact with the nascent primer strand, as shown in
Each 6G12 mutation has been reverted back to TgoT to determine the contribution of each mutation to its HNA synthetase activity. Two of the tested back mutations showed improved HNA incorporation activity, the remaining eleven mutations tested yielded small decreases in activity but retained activity above TgoT levels.
In terms of activity, 6G12 also outperforms all other polymerases isolated to date and has allowed synthesis of a longer tRNA template, even in the absence of Mn+2 ions, which is known to increase polymerase activity (at some fidelity cost). The difference in activity is shown in
HNA was typically synthesised with purified 6G12 in 100 μl reactions containing 100 pmol labelled primer, excess template (2:1), modified nucleotides and 0.5 mM manganese in reaction buffer (ThermoPol buffer—NEB). Reactions without polymerase were heated and cooled to allow primer annealing, polymerase was added and reactions were then carried out as a 3-cycle PCR (90 min at 50° C., 90 min at 65° C., 1 min at 94° C.).
Example 6a: Fidelity of 6G12 HNA SynthesisWe probed the fidelity of HNA synthesis by 6G12 by performing extension reactions lacking each one of the hNTPs. No full-length product was obtained unless all 4 hNTPs were present, as shown in
Further indications of 6G12 fidelity derive from the ELISA screen (Example 1) by which 6G12 was ultimately isolated. It involves synthesis of an HNA polymer and detection thereof by hybridization of a complementary oligonucleotide probe. Detection in ELISA is thus dependent on the accurate synthesis of the desired polymer.
A more stringent and quantitative measure of 6G12 fidelity has been obtained in conjunction with 521L (the HNA RT) suggesting that the aggregate error rate (i.e., HNA synthesis with 6G12, followed by 521L DNA synthesis from HNA, followed by amplification of the recovered DNA with Taq) to be <7×103 errors per base pair.
The following examples pertain to the isolation of a processive RNA polymerase from the CST selection experiments aimed at isolating (a) processive HNA and/or CeNA polymerase(s).
Example 7: Mutation of the Steric Gate Residue Y409 in D4 Yields a Processive, High-Fidelity RNA PolymeraseD4 is derived from Tgo, the replicative DNA polymerase from the hyperthermophillic archaeon Thermococcus gorgonarius. The starting gene (TgoT) bore mutations to disable read-ahead stalling (V93Q) and the exonuclease domain (D141A, E143A) and the Therminator mutation (A485L) to enhance incorporation of unnatural substrates. Critically, it also comprises a cluster of 8 mutations in a region of the thumb domain (aa residues 586-773) (motif 10A) and which appears critical for processivity with unnatural substrates.
The steric gate residue in D4 is still intact, however, and greatly hinders the incorporation of rNTPs. In order to improve RNA polymerase activity, the steric gate (Y409 in Tgo), was mutated and various mutants investigated (
(c) Comparison of DNA and RNA primer extension by D4N3. Reaction carried out as indicated in methods. (d) Primer extensions with complete rNTP substitution demonstrating the effect of modifying the steric gate in D4. Mutation of the steric gate residue to asparagine (Y409N) yielded D4N3, a mutant polymerase with a striking ability to processively synthesize RNAs up to 87 nt, including e.g., yeast Y-tRNA (
Following the observation that RNA polymerases often require higher Mg2+ and NTP concentrations than DNA polymerases for optimal activity, I systematically varied both rNTP and Mg2+ concentrations. The optimized conditions significantly improved processivity and speed of RNA synthesis by D4N3 as well as product yields, with D4N3 now capable of synthesising an 87 mer tRNA <20 min (
A critical parameter of polymerase function is fidelity. To investigate if D4N3 had accurately copied the DNA template strand into RNA, I cloned D4N3 transcripts by polyA-tailing and RT-PCR (Superscript II) using standard methods. Sequencing revealed a aggregate fidelity (comprising RNA synthesis by D4N3, reverse transcription by Superscript II and 30 cycles of PCR by Platinum Taq polymerase) with only 4 point mutations and 7 deletions across 19 reads of >69 bases, making a total of 13 errors in >1300 incorporations or an aggregate error rate of approximately 10−2.
Example 8: Different Mutations of the D4 Steric Gate Enable Incorporation of Other 2′-Modified NTPs2′ modified nucleotides occur widely in natural RNAs and are essential for some functions (e.g., ribosome function, thermostability of thermophile rRNA). They also have a number of interesting biotechnological applications including in nucleic acid therapeutics such as RNAi and aptamers, where they enhance potency and serum stability. However, many are poor substrates for naturally occurring RNA polymerases.
We performed a second round of modelling of the D4N3 active site, which suggested alanine, glycine or serine as optimal to accommodate bulkier 2′modifications (e.g., O-Methyl).
The smaller alanine and glycine mutations indeed allowed improved incorporation of 2′amino-ATP, 2′ara-ATP, 2′azido-ATP and 2-O-Methyl-ATP (
The complete CeTempN template (57 incorporations, including 13As) was synthesized with 2′Ara-ATP and GTP, CTP, TTP.
This suggests D4N3 and its derivatives may have improved ability to incorporate 2′ modified NTPs (notably arabino-derivatives) into RNA (or DNA).
Example 9: Only a Single Point Mutation in Motif 10A is Necessary for D4N3 ProcessivityAs only modifying the steric gate does not yield a processive RNA polymerase, we concluded that mutations in the 10A motif must be critical for processive RNA synthesis.
D4 has 8 mutations compared to wild-type Tgo in motif 10A and a sporadic L to P mutation in the A motif. Individual point mutations to revert each of the 8 mutated positions in the 10A region to wild type had little effect on RNA synthesis with the exception of position 664, reversion of which resulted in a dramatic drop in activity (
YtRNA was synthesised using purified TNQ (TgoT/Y409N/E664Q) in the presence of NEB ThermoPol buffer supplemented with an extra 2 mM MgSO4 (4 mM total) and 0.625 mM of each dNTP. 20 ml reactions contained 10 pmol FITC-labelled RNA primer, 20 pmol DNA template and 1.5 ml purified enzyme at 1:10 dilution. Cycling conditions are shown in Table 5:
Five μL of neat extensions (theoretically containing 2.5 pmol primer) were run on 13% acrylamide/8 M urea/1×TBE gel (see
This demonstrates that 2×5 minute extension cycles are sufficient to synthesise the full-length tRNA, requiring 117 rNTP incorporations, and that no product is generated in the absence of rNTPs.
The remainder of the reaction was isopropanol precipitated, pelleted and resuspended in 100 ml 1× Turbo DNase buffer containing 3 ml Turbo DNase and incubated for 90 min at 37° C. before acid phenol:chloroform extraction and a second isopropanol precipitation. This time the pellet was suspended in 100 ml dH2O, 100 ml isopropanol and 100 ml buffer RLT from a QIAGEN RNeasy kit and the RNA purified according to the manufacturer's instructions. The pure RNA was eluted in 30 ml of the provided nuclease-free water and 2 ml used for RT-PCR with both SuperScript OneStep RT-PCR System (Invitrogen) and Transcriptor One-Step RT-PCR kit (Roche).
The expected product size of 150 bp confirms the PAGE result that 2×5 min extension cycles is sufficient to synthesise the tRNA, requiring 117 rNTP incorporations.
Example 11: Synthesis of the mRNA of Protein Encoding Gene (GFP) by the Thermostable RNA Polymerase (TNQ)A GFP gene requiring 748 rNTP incorporations was synthesised using TNQ (TgoT/Y409N/E664Q) from a single-stranded DNA template made from an m6GFP-encoding plasmid. The template was prepared using one biotinylated primer and one non-biotinylated primer, such that both strands could be captured and the desired strand washed off using 0.1M NaOH. See
Synthesis was carried out in a 10 ml reaction containing 0.625 mM each rNTP, 5 pmol FITC-labelled RNA primer, 9.9 pmol ssDNA template and 0.75 ml enzyme at 1:10 dilution NEB ThermoPol buffer supplemented with 2 mM MgSO4, giving a final concentration of 4 mM Mg2+. Cycling conditions are shown in Table 6:
Following synthesis, the reactions were isopropanol precipitated and the pellet suspended in 48 ml 1× Turbo DNase buffer containing 2 ml Turbe DNase. Following a 60 min incubation at 37° C., the RNA was acid phenol:chloroform extracted and isoproponal precipitated again. The pellet was suspended in 100 ml dH2O, 100 ml isopropanol and 100 ml buffer RLT from a QIAGEN RNeasy kit and the RNA purified according to the manufacturer's instructions. The pure RNA was eluted in 30 ml of the provided nuclease-free water. A positive control reaction was run with dNTPs which was neither DNase treated nor RNeasy purified; it was purified only using a QIAGEN QIAquick PCR purification column and eluted in the same volume (30 ml) of the same water. 2.5 ml of the purified RNA was used as a template for RT-PCR using SuperScript OneStep RT-PCR System (Invitrogen) using an internal RT primer to generate a 500 bp fragment and a second primer set to generate the full-length GFP gene
A critical parameter of polymerase function is fidelity. To investigate if TNQ had accurately copied the DNA template strand into RNA and to compare its fidelity of RNA synthesis with that of D4N3 (and other RNA polymerases we cloned the full-length +rNTP and +dNTP lanes using a TOPO cloning kit (Invitrogen). A majority of the resulting colonies were visibly green, and sequencing of nine random colonies deriving from the RNA synthesis and RT-PCR confirmed an excellent aggregate fidelity (comprising RNA synthesis by D4N3, reverse transcription by Superscript II and 30 cycles of PCR by Platinum Taq polymerase) of ca. 10−3 (5 single base misincorporations and 1 insertion in 6732 sequenced bases) superior to D4N3.
Example 12: An Improved Second Gate Mutation for RNA PolymerizationFollowing the finding that only a single point mutation in the 10A region (E664Q) was necessary for processive RNA synthesis, both that position and its adjacent residues (E662, Y663, E664, Q665) were diversified individually using an NNS codon. 2×96 well plates were screened by ELISA, and limiting diversity to a single residue per experiment meant there was a low chance of missing any of the 20 possible amino acids. A typical ELISA result is shown in
Positive hits were sequenced and the following mutations, found to allow processive RNA synthesis, are shown in Table 7:
In depth investigation has not yet been carried out, but mutations at E664 have the greatest effect on RNA polymerization, with E664K appearing the most effective by a fair margin (
The compartmentalized self-transcription (CST) approach: as in CSR, compartmentalisation isolates different genotypes. A biotinylated DNA primer is used in an in vitro primer extension reaction, thus linking genotype to phenotype. Plasmid-bound primers are recovered and isolated using streptavidin-coated paramagnetic beads. Ideally, primers are too short for effective plasmid capture unless extended. Extension and processing conditions can be tailored to modulate selective pressure. Recovered plasmids are amplified and used as the starting population of subsequent rounds.
Compartmentalized self-transcription. A short biotinylated DNA primer (A) is used to select for enzymes capable of extending it in emulsion (B).
The biggest advantage of CST over CSR for selecting a CeNA synthetase is that the genetic information selected remains as DNA throughout the reaction, thus sidestepping one of the large limitations of the CSR methodology, the pull-through. Selective pressure in CST is substantially lower than in CSR and that has enabled us to carry out CST selections with full CeNTP and HNTP substitution. Enrichment estimates obtained from model experiments (w.t. vs. frame-shift mutant) suggest that it is possible to obtain a 30-fold improvement per round of selection. Some of the system's selection parameters may be optimised to further increase selection.
Protocol:
All in a 2 ml tube with a 5 mm steel bearing.
Induced library cells are prepared as in CSR. Briefly, 1 ml of culture is harvested by low speed centrifugation and resuspended in 1 ml of 1× Pol buffer. The process is repeated lowering the resuspension volume first to 500 μl and finally to 200 μl. Cell number is estimated from A595 of a 1:50 dilution. 2E8 (2×108) cells are used per selection reaction.
Emulsion is prepared in the tissuelyser using standard conditions (10″ at 15 Hz, 7″ at 17 Hz) and transferred to PCR tubes as appropriate.
Successful extension conditions for BC36N8 on a synthetic Tgo library for a first round selection with full cyclohexenyl substitution as shown in Table 9:
Reactions are then kept at 4° C. (this step may be optional).
Emulsion is broken with EBS and saturated hexanol (approximately 100 μl TBT2 and 1000 μl hexanol). Centrifuge 10′ at 13,000 RPM to separate phases. Remove 1 ml of the hexanol/oil phase, add 700 μl of hexanol, resuspend interface and re-separate by centrifugation.
DNA is recovered by isopropanol precipitation (1:10V 3 M NaOAc, 1 μl glycogen, 2V of isopropanol, overnight at −20° C.) and resuspended in 100 μl TBT2.
Twenty-five μl of the DNA solution are added to 475 μl of TBT2 and added to a Microcon YM100. Columns are spun at 2000 RPM for 40 min to filter the DNA solution. Approximately 25 μl-50 μl are recovered, and are added to pre-blocked MyOne C1 beads (10 μl per sample) in BWBS.
Add 20 μl beads to approximately 25 μl YM100-purified plasmid and add a further 400 μl of BWBS. Allow capture overnight (at least 2 hours) in overhead rotator at room temperature.
Beads are purified using the Kingfisher mL robot and the CST program (detailed in the Kingfisher mL protocol).
One μl beads can be used as template for pull-through (best done as dilution series of 20 μl reactions; around 25 cycles for a 1.8 kb product). Direct amplification of DNA from beads can also be achieved using 2 μl beads with phi29 (detailed in phi29 protocol).
Example 14: Design of Polymerase for Processive Reverse Transcription of HNA/RNAThere has been a single report to date on a B-family DNA polymerase capable of RNA reverse transcription (US2003/0228616). It reports that a mutation to the polymerase active site (A motif) on a highly conserved residue (L408) generates polymerase mutants capable of DNA synthesis from an RNA template. The mutants reported were tested (on our TgoT “wild-type” background) for HNA-RT activity unsuccessfully—no significant DNA synthesis against an HNA oligomer was detected by the polymerase activity ELISA after 2 hours extension at a range of temperatures (50° C. to 65° C.) compatible with TgoT activity.
A method has been reported to obtain information from large protein alignments based not only on sequence conservation but also on sequence co-variation: statistical coupling analysis (SCA) (Lockless & Ranganathan, Sci. 286:295 (1999)). It has been proposed that SCA can also be used to identify allosteric information transfer within proteins (Suel et al., Nat. Str. Biol. 10:59 (2003); Halabi et al., Cell 138:774 (2009)).
Working on the assumption that there are allosteric networks in polymerases and that they can affect polymerase function, we hypothesized that if there is any “information” available in the polymerase-template-primer complex regarding the nature of the template molecule, it should be possible to use SCA to identify residues near and far from the active site that correlate to polymerase function. Starting from a manually curated structural alignment of over 600 B-family DNA polymerases, SCA was used to identify potential allosteric networks within the polymerase. Unfortunately, the high conservation of residue 408 meant that it could not be included in the SCA. Because “information” must be transferred physically between a sender and receiver, we hypothesized that the residues identified by SCA in the physical vicinity of 408 could be part of an allosteric network involved in template recognition.
In that context, a shell of 5 Å around residue 408 in the 1TGO structure was selected and residues identified by SCA were selected for random mutagenesis. This approach identified residues 405, 408, 520, 521 and 575 as possible sites involved in “template information transfer”.
Initially, a conservative approach was attempted introducing a limited set of possible mutations in each of these sites (primarily hydrophobics and aromatics). Mutations were introduced by iPCR using primers coding NWC at the targeted positions. Primers were designed to have a small overlap downstream of a BsaI site. Using Roche's Expand High Fidelity according to the manufacturer's recommendations, the whole plasmid was amplified, BsaI- and DpnI-restricted and subsequently T4 DNA ligated prior to transformation.
A number of mutants were isolated and screened for HNA-RT activity using the polymerase activity ELISA screen established. Low level HNA-RT activity was detected on Y520F and L575V mutants as well as in a number of candidates mutated at position 521.
Given the results obtained, residue 521 was screened more thoroughly with mutants encoded by a primer containing NNS. Results of the subsequent screen are summarized in Table 11:
Table 11 summarizes of mutations at residue 521 identified by polymerase activity ELISA that have shown RT activity. Low HNA RT activity refers to mutants capable of incorporating at least 4 dNTP against an HNA template within the experimental time frame. High activity describes mutants that successfully synthesized a 15-mer DNA against an HNA template.
The I521L mutant was chosen for further characterisation and optimisation of reaction conditions as well as to investigate whether such mutant also had RNA-RT activity.
Although 6G12 allowed transfer of information from a DNA template into HNA, that step alone is of limited use. There have been reports on how unnatural DNA-analogue polymers (TNA and PNA) could be selected for function from forward synthesis alone (Ichida et al., J. Am. Chem. Soc. 127:2802 (2005); Brudno et al., Nat. Chem. Biol. 6:148 (2010)). There is little evidence, however, that such systems would provide a strong genotype-phenotype linkage (as fidelity information is limited) or that those moieties would allow sizeable molecules to be synthesized.
Thus, 521L is key to the process as it allows information retrieval from HNA back into DNA. As the cycle completes, it becomes possible to assess the aggregate fidelity of the two enzymes as well as begin selection for function by SELEX. Tuerk & Gold, Sci. 249:505 (1990). To prove that this was possible with our system we synthesised a tRNA molecule into HNA, using 6G12, and recovered the encoded information back into DNA, using 521L.
A number of controls and processes were introduced to minimise the possibility that the HNA synthesis step could be bypassed by the system. The key steps are summarised below:
-
- Template/primer mismatches: As previously reported (Ichida et al., Nucl. Acids Res. 33:5219 (2005)), mismatches were introduced in the template so that upon sequencing of the obtained DNA, the origin of the sequencing molecule could be determined, i.e., it should contain the primer sequences, not the original template sequence.
- Primer overhangs: Primers longer than the original template have been used to allow primer outnesting for the amplification of the recovered DNA. This increases the specificity regarding what molecules will be recovered.
- Strand-dependent reactions: Template-independent addition of DNA tails was also used to select the correct strand prior to further amplification.
- DNase and exonuclease treatments: A combination of these enzymes was used to ensure that as much as possible of the original template was degraded after 6G12 synthesis. Exonuclease was again used after 521L reaction to remove unextended RT primers that could give rise to false positive results.
- Negative controls: Reactions in which no forward synthesis (no 6G12) was carried out, reactions in which no RT (no 521L) was carried out and combinations thereof were used to ensure the recovered molecules' origins.
In addition, all reaction steps were monitored by denaturing gel electrophoresis.
A different information recovery strategy also yielded a result similar to
Tailed DNA molecules were gel purified using a denaturing gel and the DNA was successfully amplified using Superscript II RT-PCR (Invitrogen), a labeled poly-dT primer, and the same primer used to initiate RT. The Superscript (mesophilic reverse transcriptase) step was required to obtain an amplification product but, given that the enzyme does not have significant HNA-RT activity, it is believed that an enzyme capable of primer extension at low temperatures (due to the low melting temperature of the poly-dT primer) and with moderate strand displacing ability was required to generate enough DNA to be subsequently amplified by a thermostable polymerase.
The amplification product obtained was purified and TOPO-TA (Invitrogen) cloned. Isolated mutants were screened by PCR and sent for sequencing. Sequencing results showed that the (forward synthesis) primer/template mismatch was present and provided the first measure of aggregate fidelity of the two enzymes, as shown in Table 12:
Table 12 summarizes the aggregate fidelity of 6G12 DNA-dependent HNA synthesis and 521L HNA-dependent DNA synthesis. Of the 1260 bases sequenced, there were four deletions, one insertion, and the substitutions described above. In all, it suggests an aggregate error rate of less than 7×10−3/bp.
Example 16: Re-Introducing Proofreading to a Thermostable RTAs previously described, our “wild-type” enzyme has in fact four mutations over its true wild-type, including 2 mutations (D141A and E143A) which inactivate the 3′→5′ exonuclease domain, important for the proofreading function of the polymerase.
The D141A and E143 mutations, which are also present in 521L, were reverted back to their wild-type as we investigated the effect on 521L RT function. 521L mutation to 521L(exo+) was carried out by iPCR as described previously.
As expected, reintroduction of exonuclease activity reduced the observed RT activity of the enzyme for HNA. However, it improved the enzyme's RT activity on RNA. In fact, 521L(exo+) outperformed 521L on DNA synthesis from an RNA template, as shown in
Inspection of other PolB structures available, particularly the ternary complex of RB69 (Franklin et al., Cell 105:657 (2001)), suggested that the equivalent residue to I521 (L594) would be further than 5 Å from the residue equivalent to L408 (L415) at the catalytic step, when template, primer and nucleotide are all present in the active site.
This raised two possibilities within the SCA “information transfer” paradigm: either the information transfer between 408 and 521 does not occur at this step in the polymerase cycle or information is transmitted by an unidentified intermediate residue. Another clear possibility is that the SCA framework is not valid.
To test those hypotheses, the same approach that identified I521 was taken; using the I521 residue as the centre of a 5 Å shell. A number of residues were identified within that distance from I521 that had also been identified through SCA. However, to test SCA we focused our search on those residues that had not been present in SCA: Y388, G517 and T541.
A scanning approach similar to 521 was taken with mutant libraries generated by iPCR with primers containing NNS at the target positions. Individual mutants were isolated and used to screen for RT activity.
Residues G517 and T541 are highly conserved residues (>96% identity), with T541 being flanked by two aspartate residues involved in the catalytic step. As expected, screening at those positions yielded no enzymes with HNA RT activity above wild-type.
The remaining residue, Y388, is very poorly conserved which probably accounts for its absence in SCA. Interestingly, a number of different side-chains at residue 388 did display HNA RT activity: V, R, H, N, and T. These are now being further characterised to assess their potential as well as their potential in a 521L background. Residue 388 is immediately downstream of a motif reported to affect polymerase fidelity and processivity: YXGG/A (Truniger et al., EMBO J. 15:3430 (1996); Bohlke et al., Nucl. Acids. Res. 28:3910 (2000)).
In view of those results, questions are raised regarding the use of SCA predictions to identify possible improvements in RT activity. While Y388 could be a false negative not identified by the method's current implementation, it may also suggest that a simpler hypothesis is at play: Residues within a certain distance from the catalytic site (particularly the C motif central triad DTD) can be mutated to subtly alter the polymerase function, in this case HNA RT activity.
Example 18: Information Transfer from DNA to CeNA and Back from CeNA to DNASimilar to earlier examples, genetic information from a DNA template can be transferred to a CeNA molecule and recovered using the isolated polymerases.
Forward synthesis (DNA→CeNA) was carried out with C7 in very similar conditions to previously described ones for 6G12. Typical reactions were carried out in ThermoPol buffer (NEB) with 1 M primer and template and suitable CeNTP concentration (30˜500 μM of each nucleotide). Reactions could be carried out in a range of temperatures compatible with B-family thermostable polymerases. Like 6G12, C7 can also start unnatural nucleic acid synthesis from both DNA and RNA primers (
Similar to reactions with 6G12 and HNA, a number of steps (described in the corresponding example above) were introduced to ensure that no original DNA template was carried forward and to be able to distinguish any potential carry over from primer synthesized molecules.
As 521L had already shown RT activity against RNA and HNA, it was the obvious candidate to be used against CeNA. As with HNA, a DNased and purified forward synthesis was used as template for the RT reaction, shown in
DNA synthesis by 521L from a CeNA template was successful confirming 521L as a RNA, HNA and CeNA RT. A typical CeNA-RT 521L reaction (50 μl) was carried out using equal concentrations of labeled primer and CeNA template in 1× ThermoPol buffer (NEB). After annealing the primer-template complex, 521L was added and the reaction carried out as a four-cycle PCR with extension steps of 4 hours at 65° C.
Example 19: Specific Exemplary SequencesIt should be noted that the designations in this example are clone names indexed in the sequence listing, and are not indications of amino acids (e.g., C7=clone C7 and does not indicate cysteine at position 7). These sequences are shown in Table 13:
We sought to address genetic information en- and de-coding. Broadly, we asked if an artificial genetic system supporting both heredity and evolution could be constructed from entirely unnatural components. Minimally such a system requires a chemical framework (XNA) capable of crosstalk with DNA or RNA, a means for XNA synthesis (i.e., a DNA-templated XNA polymerase, to transfer natural genetic information from DNA to XNA) and means for decoding XNA (i.e., a XNA reverse transcriptase). We identified HNA (1,5 anhydrohexitol nucleic acid) and CeNA (cyclohexenyl nucleic acids) as potential XNAs with desirable physico-chemical properties (
The poor activity of even TgoT in utilizing hNTPs or ceNTPs precluded the application of established methods of polymerase engineering. Sweasy & Loeb, PNAS 90:4626 (1993); Ghadessy et al., PNAS 98:4552 (2001); Xia et al., 2002. We therefore developed a new highly sensitive selection strategy called compartmentalized self-tagging (CST) to enable the discovery of polymerases capable of processive HNA or CeNA synthesis. CST is based on a positive feedback loop, whereby a polymerase tags its own encoding gene by extension of a metastable biotinylated oligonucleotide. Extension stabilizes the oligonucleotide-plasmid complex and enables the selective capture of plasmids encoding active polymerases (
To expedite the discovery of HNA and/or CeNA polymerase activities diversified, we created 22 separate mutagenesis libraries of TgoT. Diversity comprised phylogenetic variability as well as 5% to 10% random mutations (at conserved positions) (
We first performed CST selection separately on each of the 22 TgoT libraries and scored their hNTP and ceNTP incorporation potential before and after selection by polyclonal primer extension. This screen, when mapped on the polymerase structure (
Natural nucleic acids can be decoded by the action of templated polymerases such as reverse transcriptases (RTs) in the case of RNA. Without a means of decoding HNA polymers, HNA synthesis (although potentially useful for the bulk production of ssHNA oligomers for applications in gene silencing), is a “dead-end” because genetic information transferred from DNA remains locked in HNA, precluding both analysis and evolution. However, none of the available polymerases displayed HNA RT activity. We therefore decided to develop an HNA RT de novo in the parent polymerase TgoT. Reverse transcriptase activity (from an RNA template) had been described in the related Pfu DNA polymerase, upon mutation of L409. Arezi et al., U.S. Patent Pub. No. 2003/0228616.
We hypothesized that due the HNA's propensity for an RNA-like A-form conformation, HNA-RT activity might be found in the structural neighborhood of an RNA RT. Mutation of the equivalent L408 in TgoT, however, only yielded variants with weak but detectable HNA-RT activity. We therefore chose to explore the structural and functional context of L408 (a highly conserved residue in the polB family) in more detail. We used Statistical Correlation analysis (SCA), a statistical approach to score pairwise positional correlations of sequence variation in phylogeny, to discover positional covariation in the vicinity of L408 as part of potential allosteric interaction network involved in template recognition. SCA has been suggested to allow inference of functional amino acid networks and had previously been applied to rationalize selected mutations (Loakes et al., J. Am. Chem. Soc. 131:14827 (2009); Lockless & Muir, PNAS 106:10999 (200)) and aid protein design (Russ et al., Nat. 437:579 (2005)). Sequence variation within the PolB family genes deposited in GenBank was found to be too high to provide sufficiently accurate alignments for SCA. We therefore compiled a hand-curated dataset of 671 non-redundant PolB sequences based on structural alignment as input for SCA. Random mutation of SCA hits (F405, Y520, I521, L575) and PAA screening of the four mini-repertoires identified TgoT: I521L (RT521), as a general and processive HNA RT as demonstrated by the reverse transcription of tHNA supE (see above) into DNA (
The ability to both synthesize and reverse transcribe HNA allowed us to determine the fidelity of information transfer between DNA and HNA. We determined aggregate error rates (the sum of error rates of both HNA synthesis and HNA reverse transcription) by cloning and sequencing of E. coli tRNA supE converted into HNA by Pol6G12 and back into DNA by RT521. Correcting for the contribution of 30 cycles of PCR we obtained an aggregate misincorporation rate of 8.3×10−3 and aggregate indel (insertion/deletion) rates of 5.2×10−3. Deconvolution of individual error rates (estimating RT521 fidelity from reverse transcription of an identical RNA template) yields a fidelity of 8×10−3 for Pol6G12 (and 1.2×10−3 for RT521) (
Inspection of the aggregate mutation spectrum reveals predominantly A/G, C/T transition mutations commonly observed for all polymerases due to base tautomerism. We also observe an uncommon preponderance for T/G transversions, however, indicative of hG misincorporation opposite template dT by Pol6G12 (or dA opposite template hC by RT521).
Together the HNA polymerase Pol6G12 and RT521 HNA RT establish a synthetic genetic system built on HNA-based heredity.
Example 21: Application of Invention to HNA AptamersWe asked if such a HNA based synthetic genetic system might also support Darwinian evolution. We therefore initiated in vitro selection experiments for HNA aptamers. The aggregate fidelity of information transfer between DNA and HNA and back should be readily be compatible with selection of HNA sequences >100 nts in length24 but the capacity of ssHNA oligomers to fold into stable three-dimensional structures capable of specific recognition of molecular targets was unknown.
We therefore first selected HNA aptamers against a well characterized nucleic acid target, the HIV TAR (trans-activating response) RNA for which both DNA and RNA aptamers had previously been isolated. Boiziau et al., J. Biol. Chem. 274:12730 (1999); Duconge & Toulme, RNA 5:1605 (1999). HNA TAR binders evolved readily but, unlike RNA or DNA aptamers, did not appear to bind TAR via a “kissing loop” interaction. Mapping the interaction of HNA anti-TAR aptamers revealed two clades targeting either the TAR-stem and asymmetric bulge or requiring both loop and bulge for binding (
The synthetic genetic system described herein provides a synthetic route to a new sequence space not previously accessible for exploration. The invention may be used to investigate how this “HNA space” is populated with novel phenotypes; it appears that at least HNA aptamers displaying specific ligand binding are readily discovered. If selections are generalized to a wider range of targets, such HNA aptamers may have great biotechnological and therapeutic potential due to the non-cognate chemical makeup providing robust chemical stability and low toxicity as both nucleosides and oligomers.
Such synthetic genetic systems should provide a rich source of new receptors and catalysts with tailor-made chemistries for applications in diagnosis and therapy.
Detailed Description of Drawings for Examples 20-21(a) Pol6G12 mutations mapped to structurally equivalent E. coli pol II (3MAQ) residues. Eleven of the 14 mutations identified in Pol6G12, shown in red, can be mapped to the pol II ternary complex, with nearly half of those clustering in the vicinity of the nascent HNA strand, shown in green.
(b) Purified wild-type enzyme does not synthesise HNA much beyond six incorporations, but purified Pol6G12 can quantitatively synthesise HNA, such as the E. coli amber suppressor tRNA gene shown. (c) Single-stranded HNA is refractory to all nucleases tested and substantially more resistant than DNA in acidic environments (d). Half-life of HNA under those conditions (t1/2HNA=347 min, R2=0.899) is nearly eight-times higher than DNA (t1/2DNA=43.3 min, R2=0.975).
(a) A hand-curated sequence alignment of 671 non-redundant B-family polymerases was used in SCA to identify pairs of co-varying residues to identify allosteric networks within the polymerase. The distribution of covariation values obtained fitted a log-normal distribution (μ=−1.749, σ=0.808). Values above the 99th percentile (kT*>1.964) were considered significant and used to establish a PolB allosteric network. Highly significant residues (kT*>2.4) are shown mapped onto the Tgo (1TGO) apo structure together with conserved residues that could not be included in SCA). (b) SCA results shown in the related E. coli pol II (3MAQ)—SCA and conserved residues are shown as in (a), primer strand and template. (c) Hierarchical clustering of the residues identified to co-vary with I521 in the alignment used.
Base composition, particularly purine and pyrimidine bias, has a strong influence on HNA/DNA melting temperature (Boudou et al., Nucl. Acids Res. 27(6):1450 (1999)). By making use of these biases, it is possible to design a DNA primer capable of binding HNA and to be successfully extended by 521-type polymerases (HNA RT).
Initially, three DNA primer sequences were tested against a known HNA molecule (to be synthesised from a DNA template (Testbind3), as shown in Table 14:
A NAPfd (previously described; binding site shown underlined in Table 14) 2′OMe RNA primer is used to synthesise an HNA molecule that harbours the binding site for all three test probes. The synthesised HNA was isolated by PAGE-purifying a TURBO DNAsed synthesis reaction (as described) and primer binding experiments carried out in a variety of conditions, as shown in
Having proved that a DNA primer could bind a compositionally-biased HNA molecule, I repeated the experiment for test7 on an unbiased template (TempNpurine:
with similar results. DNA primer binding to HNA was unaffected by a 5′ overhang on the DNA.
Having obtained successful primer binding, we carried out a primer-dependent RT as a proof-of-principle.
Having obtained an adequate RT product, we investigated the limit of detection of the system in a molecule of known sequence (TempNpurine). PCR detection of the RT product (using a double outnest approach) suggested RT521 could detect HNA RT products down to RTs carried out with 0.25 pmol HNA.
Further primer optimisation, including spiking of the DNA primer with other nucleotide chemistries (e.g., LNA, 2′OMe-RNA) is being carried out. An alternative, however, was to further improve the RT enzyme itself.
Example 23: Further Design of Polymerases for Processive Reverse Transcription of HNA/RNAThe E664K mutation, described in the context of designing an RNA polymerase, was determined to increase the affinity of the polymerase for the primer-template complex. Because an HNA/DNA hybrid is expected to give rise to a substantially distorted helix, increasing the affinity of the RT521 for this template is likely to improve HNA reverse transcription.
iPCR was used to introduce the E664K mutation in the RT521 context and both enzymes were tested side-by-side for HNA RT-PCR using the TempNpurine system described above.
Carrying out a similar experiment using an in-nested PCR to increase the sensitivity of detection yielded clear amplification of a single HNA sequence down to 9 attomoles of input HNA into the RT step carried out with 521K. This is not expected to be the limit of detection—further reaction optimisation would be expected to further improve sensitivity.
Importantly, Test7 is portable and can be used against other templates, including degenerate libraries such as ApLib5
shown in
DNA polymerase substrate specificity is critical for genomic integrity; the current paradigm is that it is exclusively determined by the active site, as exemplified by steric gate mutations. Such mutations increase incorporation of NTPs by DNA polymerases from all families by several orders of magnitude but do not result in processive RNA polymerases. Indeed, most modified DNA polymerases stall after 6-7 NTP incorporations, despite impressive incorporation efficiency. Through a focus on the thumb domain of a B-family polymerase, Tgo, we have identified a point mutation in the immediate vicinity of the nascent strand which relieves this extension block: the “second gate”. Mutation of the second gate of Tgo (E664K), combined with a classical steric gate mutation (Y409G) results in TGK: the first primer-dependent, thermostable RNA polymerase engineered from a DNA scaffold. TGK can synthesise a tRNA gene in less than a minute and can synthesise a 1.7 kb Luciferase gene in only 1 hour. Furthermore, the E664K mutation enables translesion synthesis even in the absence of the steric gate mutation. The “second gate” thus specifies the critical missing step in the evolutionary path from DNA to RNA polymerase, defines a new post-synthetic determinant of polymerase substrate specificity distal to the active site and establishes the thumb domain as region that merits further investigation in studies of both replication fidelity and polymerase substrate expansion.
DNA polymerase substrate specificity is of central importance for genome stability, as well as for applications in biotechnology and is generally assumed to be determined solely by the geometry and chemistry of the polymerase active site. Yet, highly efficient incorporation of nucleotide substrates does not necessarily beget processive synthesis of nucleic acid polymers, as is exemplified by mutation of the “steric gate” residue in the active site of the DNA polymerases. Such mutations increase the catalytic efficiency of NTP incorporation by several orders of magnitude, but the resulting polymerases still remain incapable of processive RNA synthesis. Here we describe the discovery of a critical secondary specificity determinant, a “second gate,” located in the polymerase thumb domain, 25 Å from the primer 3′ terminus. Mutation of this second gate residue (E664K) in Tgo, the replicative DNA polymerase from T. gorgonarius, together with a classic steric gate mutation (Y409G), yields a processive, thermostable, primer-dependent RNA polymerase, capable of synthesizing protein-coding mRNAs up to 1.7 kb in length with a fidelity comparable to T7 RNA polymerase. The “second gate” specifies the critical missing step in the evolutionary path from DNA to RNA polymerase and defines a new post-synthetic determinant of polymerase substrate specificity distal to the active site.
Despite significant progress in understanding polymerase substrate specificity, the engineering of a processive RNA polymerase from a DNA polymerase scaffold has proved elusive. Mutation of the “steric gate” residue in the active site of the DNA polymerases increases the catalytic efficiency of NTP incorporation, but the resulting polymerases remain incapable of processive RNA synthesis. Here, we describe the discovery of a critical secondary determinant, a “second gate”, located in the polymerase thumb domain, 25 Å from the primer 3′ terminus. Mutation of this second gate residue (E664K) in Tgo, the replicative DNA polymerase from T. gorgonarius, together with a classic steric gate mutation (Y409G), yields the first processive, thermostable, primer-dependent RNA polymerase, capable of synthesizing protein-coding mRNAs up to 1.7 kb in length with fidelity comparable to T7 RNA polymerase. This “second gate” specifies the critical missing step in the evolutionary path from DNA to RNA polymerase and defines a new post-synthetic determinant of polymerase substrate specificity distal to the active site.
DNA polymerases enable the propagation of genetic information through faithful replication and maintenance of the genome and thus are of central importance to all life. Genome replication requires sophisticated substrate recognition mechanisms to ensure polymerase fidelity and to exclude non-cognate and/or damaged nucleotides from incorporation into the genome. Detailed structural investigations together with nucleotide chemistry have begun to reveal the molecular mechanisms of how the polymerase active site may distinguish cognate from non-cognate nucleotide chemistries and geometries. Of particular importance for the integrity of the DNA-based genomes is the exclusion of ribonucleotides from incorporation into the genome. Ribonucleotide triphosphates (NTPs) differ from the deoxyribonucleotide triphosphates (dNTPs) only by the presence of a 2′-hydroxyl (—OH) on the ribofuranose ring and are present in the cell at concentrations up to 100-fold in excess of the cognate dNTPs. Although DNA polymerases exclude NTPs from their active site and hence from the genome it has recently been shown that incorporation does occur to a detectable degree, with significant implications for genome stability and repair. This issue is even more acute for thermophillic organisms as high temperatures would further increase genome instability by accelerating the spontaneous degradation of RNA by nucleophilic attack of the vicinal 2′ OH of ribose on the 3′ phosphodiester bond.
Consequently, DNA polymerases use an exceptionally effective mechanism to prevent NTP incorporation into the nascent strand; a single residue, the “steric gate”, exerts stringent steric control of the chemical nature of the 2′ position of the incoming nucleotide. Steric gate residues have been identified in all replicative (polB, polC) (Evans et al., PNAS 105:20695 (2008); Bonnin et al., J. Mol. Biol. 290:241 (1999); Yang et al., Biochem. 41:10256 (2002); Gardner & Jack, 1999) and repair (polA, polY, polX) (Astatke et al., PNAS 95:3402 (1998a); DeLucia et al., Nucl. Acids. Res. 31:4129 (2003); Jarosz et al., Nat. 439:225 (2006); Brown et al., J. Mol. Biol. 395:282 (2010)) (with the possible exception of Pol Mu (Ruiz et al., Nucl. Acids. Res. 31:4441 (2003)) polymerase families and the reverse transcriptases (Gao et al., PNAS 94:407 (1997); Cases-Gonzalez et al., J. Biol. Chem. 275:19759 (2000); Entin-Meer et al., Biochem. J. 367:381 (2002); Boyer et al., PNAS 97:3056 (2000)) (RTs,
Although mutation of the steric gate residue commonly renders a DNA polymerase permissive for NTP incorporation, such mutations do not by themselves enable synthesis of extended RNA oligomers. The engineering of an RNA polymerase from a DNA polymerase has been attempted using rational design (Yang et al., 2002), in vitro or in vivo screening (Patel & Loeb, J. Biol. Chem. 275:40266 (2000); Staiger & Marx, Chembiochem 11:1963 (2010)) and directed evolution by phage display (Xia et al., PNAS 99:6597 (2002)) or compartmentalized self-replication (Ong et al., 2006). Although these efforts have yielded DNA polymerases that efficiently incorporate NTPs, most stall at n+6 and none are able to synthesize RNA oligomers longer than 58 nucleotide incorporations and even this typically requires long incubation times (several hours), high polymerase concentrations and mutagenic metal ions (Mn2+). Patel et al., 2000; Staiger & Marx, 2010; McCullum & Chaput, Chem. Commun. 20:2938 (2009); Shinkai et al., J. Biol. Chem. 276:18836 (2001).
Thus we (Ong et al., 2006) and others (Astatke et al., 1998a; Brown & Suo, 2011) have reasoned that, apart from the steric gate residue in the active site, there must be at least one other critical determinant in the DNA polymerase structure that aborts processive RNA synthesis. Indeed, evolution of a processive RNA polymerase from a DNA polymerase framework must be possible, as there is a natural precedent. Structural and phylogenetic evidence suggest that the single subunit RNA polymerases (ssRNAP) of mitochondria and T-odd bacteriophages, to which the widely used T7 RNA polymerase belongs, derive from an ancestral A-family DNA polymerase. Delarue et al., Prot. Engin. 3:461 (1990); Moras, Nat. 364:572 (1993); Sousa, Trends Biochem. Sci. 21:186 (1996); Cermakian et al., J. Mol. Evol. 45:671 (1997). Although the polymerases from the two families have diverged widely and there is no known extant “missing link,” there must be an adaptive path linking RNA polymerases and DNA polymerases.
Here, we describe the discovery and characterisation of a critical determinant of polymerase substrate recognition located in the thumb domain of T. gorgonarius DNA polymerase (Tgo), 25 Å from the primer 3′ terminus (
All DNA oligonucleotides were from Sigma, IDT, Eurogentech or MWG Eurofins and all RNA oligonucleotides were from Dharmacon or IDT. All dNTPs used were from Roche (Roche Diagnostics GmbH, Germany), GE (GE Healthcare Life Sciences, UK) or Agilent (Agilent Technologies Inc, California, USA). All NTPs used were from Roche., 2′fluoro and 2′azido dNTPs were from (TriLink Biotechnologies Inc, California, USA) and 2′iodo-dATP was from Jena Bioscience (Jena Bioscience GmbH, Germany).
DNA Manipulation, Protein Expression and PurificationAll DNA manipulation and small-scale expression was carried out in NEB 10-beta cells (New England Biolabs Inc., Massachusetts, USA). TgoT and all mutants were maintained in pASK75. Large scale expression and purification was as described (Ramsay et al, 2010) except that either BL21 CodonPlus-RIL (Agilent Technologies) or NEB T7 Express LysY (NEB) were used; cultures were induced for 4 hours at 37° C. and cleared lysates were pre-cleaned on DE52 anion exchange resin (Whatman Inc, New Jersey, USA) prior to loading onto the 6/10 Hi-Prep Heparin FF column. All polymerases eluted at 0.7-0.8 M NaCl, were filter dialyzed (Amicon Ultra Centrifugal Filters 50K; Millipore, Mass., USA) into 2× Vent storage buffer (NEB) and stored in 50% glycerol. Mutants were typically expressed from NEB 10-beta cells (NEB), lysed by heat treatment and stored in 1× Thermopol buffer (NEB) and screened for activity as cleared 10× lysates.
Point mutations were introduced by iPCR using either Expand Hi-Fidelity PCR System (Roche) or Herculase II (Agilent Technologies), BsaI (NEB) digested, ligated with T4 DNA Ligase (NEB) and sequenced to confirm the presence of mutation prior to expression, except L403P which was introduced to TgoT by cutting TgoT and D4 with XbaI and Bsu36I, gel purifying the digests and ligating the appropriate fragments to generate TgoT L403P and D4 P403L.
Compartmentalised Self-Tagging (CST)D4 was isolated from the first round of CST selection.
Primer ExtensionPrimer extension for screening (either for mutant polymerases or for activity with novel substrates) was carried in 3-5 μl reactions containing 1-10 pmol primer with template at 2-fold excess. Usually, primer FD (5′-CCCCTTATTAGCGTTTGCCA-3′) (SEQ ID NO:53) in either DNA or RNA with 5′biotin, 5′FITC, 5′Cy3, 5′Cy5 or dual-labelled with 5′FITC-dT-biotin was used to extend TempN (5′-CTCACGATGCTGGACCAGATAAGCACTTAGCCACGTAGTGCTGTTCGGTAATCGAT CTGGCAAACGCTAATAAGGGG-3′) (SEQ ID NO:54) in 1× Thermopol buffer (NEB) with 0.25-0.75 mM each NTP and supplemented with MgSO4 in the case of certain steric gate mutants. A typical extension protocol was 2 cycles of 10 sec 94° C., 1 min 50° C., 10 min 65° C.
For time courses, 2 pmol RNA primer YtRHNA2HNA2
underlined section is template binding site) was annealed to 4 pmol template tRNAtemp1 (5′-GGTGGGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATCTGCCGTCATCGACTT CGAAGGTTCGAATCCTTCCCCCACCACCA-3′, based on GI: 174470) (SEQ ID NO:56) per reaction in 1× Thermopol, +3 mM MgSO4, 2.5 mM NTP (0.75 mM each NTP), by heating to 94° C. for 30 sec and cooling to 4° C. at 0.1° C./sec. Enzyme was added on ice and the reactions incubated at 65° C. prior to quenching with 2V 98% formamide/10 mM EDTA and separating on 15% acrylamide/8M urea PAGE.
The polymerase activity assay (PAA) screen was carried out as described (VP, CC, PH, manuscript in preparation), except 10 μl 8M urea/10 mM EDTA containing the digoxygenin (DIG)-labelled probe oligonucleotide was added to the reaction mix (as for primer extension, typically 5-μl reactions containing 5 pmol 5′biotin-FD and 10 pmol TempN) following primer extension using biotinylated primer and incubated at 65° C. for 2 min. This hot mixture was added to 200 μl pre-chilled PBS-T (PBS+0.1% Tween-20) in a 96-well StreptaWell plate (Roche) and the probe allowed to hybridise to the extended primer while the biotin bound to the plate. The plate was washed three times by immersion in a PBS-T bath and the probe was detected using an anti-digoxygenin-POD Fab fragment (Roche) and Ultra-TMB ELISA substrate (Thermo Scientific, Massachusetts, USA).
Protein Concentration AssayPurified protein concentration was assayed via separation on NuPAGE 4-12% Bis-Tris gels (Invitrogen Ltd, UK), staining with SYPRO orange (Sigma-Aldrich, Missouri, USA) and quantification by Typhoon TRIO and ImageQuant. A standard curve generated from BSA standards of known concentration (Thermo) was used to derive polymerase concentration.
GFP and Luciferase Template Preparation and SynthesisTemplates were prepared from mGFP6 (Haseloff, Meth. Cell Biol. 58:139 (1999) for GFP or Luciferase T7 Control DNA (Promega #L482A) by PCR using Herculase II (Agilent Technologies) with one biotinylated primer and one non-biotinylated primer. This method allowed the introduction of a unique forward priming site and allowed generation of single-strand DNA templates by binding the QIAquick (Qiagen NV, Netherlands)-purified PCR products to an appropriate volume of paramagnetic beads (DynaBeads MyONE Streptavidin C1, Invitrogen). The desired strand was eluted using 20 mM or 100 mM NaOH at 37° C., neutralised in an equal volume of 3M NaOAc pH5.5 and isopropanol precipitated.
Forward synthesis was from RNA primer L3T32
underlined is template binding site, 5′ of sequence introduces outnest for PCR) in a 50 μl reaction consisting 1× Thermopol (NEB), +3 mM MgSO4, 2.5 mM NTP (0.75 mM each), with a 1:1 primer:template ratio varying from 10 pmol to 70 pmol of each and 150 nM TGK. Extension was two cycles of 10 sec 94° C., 1 min 50° C., 1 hr 65° C., to make 2 hr total extension. The DNA template was digested by TURBO DNase (Applied Biosystems/Ambion, Texas, USA) treatment (8 U for 1 hr at 37° C. in reaction mix supplemented with 1× TURBO DNase buffer) and RNA purified on an RNeasy column (Qiagen).
Reverse transcription of purified RNA was carried out using Transcriptor RT (Roche) with primer GB1lucfo
underlined is template binding site, 5′ of sequence introduces outnest for PCR) for luciferase or
underlined is template binding site, 5′ of sequence introduces outnest for PCR) and cDNA PCR-amPpifie using FastStart Taq (Roche) with primers LMB3+ (5′-CAGGAAACAGCTATGACAAA-3′) (SEQ ID NO:60) and NAP (5′-CAGTATCGACAAAGGA-3′) (SEQ ID NO:61). PCR products were TOPO TA-cloned into pCR4.1 using TOPO TA Cloning Kit For Sequencing (Invitrogen) and sequenced from colonies. Both forward synthesis and RT primers introduced mismatches so that sequences could be identified as having been both synthesized and reverse transcribed. Error rates were calculated using the Transcriptor error rate provided by Roche (1.98×10−5) and assuming a Taq error rate.
ssDNA templates were described as above, except using primer L3SDGFPba (5′biotin-CAGGAAACAGCTATGACAGGAGGAGCGAGATGAGTAAAGGAGAA GAACTTTTC-3′) (SEQ ID NO:62) which encodes a Shine Dalgarno sequence (underlined). Primer RNA L3AGG (5′-CAGGAAACAGCTATGACAGG-3′) (SEQ ID NO:63) was used for RNA synthesis as described above for 2 cycles of 10 sec 94° C., 1 min 50° C., 15 min 65° C. to make 30 min total extension and purified as previously described. IVT was carried out using 1.4 μg RNA added directly to PURExpress In Vitro Protein Synthesis Kit (NEB) supplemented with 3 μl FluoroText GreenLys in vitro Translation Labelling System (Promega) and incubated for 2 hr at 37° C. prior to analysis on a NuPAGE Novex 10% Bis-Tris Gel (Invitrogen).
Electrophoretic mobility shift assay (EMSA)
The affinity of mutant Tgo polymerases for DNA:DNA or DNA:RNA was assayed using 5′FITC-labelled primer K0 (5′-GCACGGCAGCACGTG-3′) (SEQ ID NO:64) in either DNA or RNA and template TempK4 (5′biotin-ACTGCGATGACTGTACTCGTCTAGTAGCACTGCACGTGCTGCCGTGC-3′) (SEQ ID NO:65) in a 5 μl reaction mixture containing 1× Thermpol buffer (NEB), 250 fmol primer K0, 2.5 nmol TempK4, 1 mM EDTA, 1U RNasin (Promega) and 0.02-1.25 μM polymerase. The reactions were mixed, heated to 94° C. for 30 sec and cooled at 0.1° C./sec to 4° C. before loading onto precooled 6% TBE gel (Invitrogen) and run for 1 hr at 100V in precooled 1×TBE. Bands were quantified using a Typhoon Trio and ImageQuant and the shift fitted to y=(Bmax*x)/(Kd+x) using MatLab (The MathWorks Ltd).
Polymerase Processivity and Lesion Bypass AssaysThe 48-mer DNA templates, 5′-TCG-ATA-CTG-GTA-CTA-ATG-ATT-AAC-GAA-YXA-AGC-ACG-TCC-GTA-CCA-TCG-3′, where YX=TT, TT-CPD (cis-syn cyclobutane pyrimidine dimer), or TAb (Ab-abasic site) (SEQ ID NO:66), and 16-mer DNA primer (5′-TGG-TAC-GGA-CGT-GCT-T-3′) (SEQ ID NO:67), or RNA primer (5′-UGG-UAC-GGA-CGU-GCU-U-3′) (SEQ ID NO:70) were used in the primer extension experiments. Primers, undamaged, and abasic site-containing templates were synthesized by Lofstrand Laboratories (Gaithersburg, Md.). The CPD-containing template was synthesized by Phoenix Biotechnologies (Huntsville, Ala.). All oligonucleotides were gel purified prior to use. Primers were 5′-end-labelled using T4 polynucleotide kinase and [γ-32P]-ATP. DNA substrates were prepared by annealing DNA templates with 32P-labelled primers at a 1.5:1 molar ratio. Hybridization was achieved by heating the required mixture in an annealing buffer (50 mM Tris-HCl (pH 8), 5 mM MgCl2, 50 μg ml−1 BSA, 1.42 mM 2-mercaptoethanol) for 10 min at 100° C. followed by slow cooling to room temperature over a period of about 2 hr. Annealing efficiencies were >95%.
Standard reactions were performed for 4 min at 65° C. in 1× Thermopol buffer and contained 10 nM DNA templates (expressed as primer termini), 0.25 mM of dNTPs or NTPs mixtures and appropriate nM TgoT, TGE, TYK, or TGK. To analyze enzyme processivity, reactions were performed with a large molar excess of DNA template over the polymerase ensuring “single-hit” conditions. The reactions were terminated by mixing with 1 vol. of formamide loading dye solution containing 50 mM EDTA, 0.1% xylene cyanol and 0.1% bromophenol blue in 90% formamide. Before loading onto the gel, the reactions were denatured by heating at 100° C. for 10 min and immediately transferred onto ice for 2 min. Products were resolved by denaturing polyacrylamide gel electrophoresis (8 M urea, 15% acrylamide, 3 hr at 2000 V) and then visualized and quantified using a Fuji image analyser FLA-3000 and MultiGauge software. Termination probabilities (expressed as a percentage) for template positions 1-14 were calculated as the band intensity at the specific position divided by the intensity at that position plus all longer products. Kokoska et al., J. Biol. Chem. 278:50537 (2003).
Identification of a Polymerase Region Enabling Improved RNA SynthesisPolymerases capable of synthesizing novel unnatural nucleic acid polymers are described. Some of these, in which the canonical ribofuranose ring is replaced by an alternative structure, for example a six-membered 1,5 anhydrohexitol ring (hexitol nucleic acids, HNA), display A-like (RNA-like) helical conformations (Herdewjin, Chem. Biodivers. 7:1 (2010); Lescrinier et al., 7:719 (2000)) (
D4 derives from a variant of the replicative DNA polymerase of the hyperthermophillic archaeon Thermococcus gorgonarius (Tgo), which bears additional mutations to disable uracil stalling (V93Q) (Fogg et al., Nat. Str. Biol. 9:922 (2002)) and 3′-5′ exonuclease activity (D141A, E143A) as well as the “Therminator” (Gardner & Jack, 2002) mutation (A485L) to enhance incorporation of unnatural substrates. This mutant polymerase (henceforth termed TgoT) does not display RNA polymerase activity above background levels: RNA synthesis by TgoT stalls after 6-7 incorporations from a DNA primer and is absent from an RNA primer. In contrast, D4 extends both DNA and RNA primers by >20 nts, under the same conditions (
We aimed to better understand the function of these mutations to the RNA polymerase phenotype, especially of the eight mutations distal to the active site. In order to determine their contribution to RNA polymerase activity in the context of a more permissive active site we introduced a steric gate mutation into D4. Previous work on the B family (polB) polymerases had identified a conserved tyrosine (Tgo: Y409) as the steric gate residue. Replacing Y with a smaller residue is known to reduce NTP/dNTP discrimination by >103-fold, yet not to enable processive RNA synthesis. Bonnin et al., 1999; Yang et al., 2002; Gardner & Jack, 1999. Y409 is also thought to be involved in metal ion coordination and productive nucleotide positioning, however, and we were concerned that a “null” mutation (e.g., to a small side-chain amino acid, such as A) might adversely affect overall polymerase function and fidelity. Blasco et al., J. Biol. Chem. 267:19427 (1992); Yang et al., Mol. Cell 22:5 (2006); Kirouac et al., J. Mol. Biol. 407:382 (2011).
Because no archaeal B-family polymerases have been crystallised as a ternary complex to date, we carried out in silico modelling using the homologous polB RB69 (PDB: 1Q9Y) (Freisinger et al., EMBO J. 23:1494 (2004)) to find an amino acid side chain that would relieve the steric clash with the incoming NTP while maintaining sufficient bulk to avoid relaxing the geometry of the active site. We found that replacement of Y with medium sized side-chains (e.g., S, L, N) improved RNA polymerase activity in a D4 background. Indeed, introduction of Y409N mutation into the D4 context (D4: Y409N, henceforth called D4N) yielded a highly proficient RNA polymerase capable of synthesizing a tRNA (E. coli supF tRNATyr) gene consisting 65 nucleotide incorporations in 20 minutes, while the same mutation introduced into the “wild-type” polymerase TgoT (TgoT: Y409N, TN) only marginally improved RNA polymerase activity (
Having established that the nine mutations in D4 in conjunction with the Y409N steric gate mutation enabled processive RNA synthesis, we sought to determine the contributions of the different mutations in D4N to RNA synthesis. To this end, we reverted each individual D4N mutation to wild-type and determined the effect on RNA synthesis. Reversion of the thumb domain mutations revealed a striking pattern: seven of the eight back mutations (D4N: T657P, Q658E, H659K, H663Y, A669D, K671N, T676I) did not reduce RNA polymerase activity, whereas reversion of one specific residue to wild type (D4N: Q664E) had a dramatic negative effect on processive RNA synthesis (
To determine the effect of this key mutation free of the context of the other D4 mutations, we introduced the E664Q forward mutation together with the steric gate mutation Y409N de novo into the TgoT framework. The resulting TgoT polymerase double mutant TNQ (TgoT: Y409N, E664Q) displayed superior RNA polymerase activity compared to both the steric gate mutant TN (TgoT Y409N) and the original D4N polymerase. These results indicate that the E664Q mutation is both necessary and sufficient for processive RNA synthesis. As DNA polymerases are incapable of processive RNA synthesis even when the steric gate is mutated (as shown previously (Astatke et al., 1998a; Patel et al., 2000; Staiger & Marx, 2010; Xia et al., 2002; Ong et al. 2006) and here in the example of TN), we conclude that E664 forms (or is a critical part of) a second checkpoint in the DNA polymerase structure that normally prevents the synthesis of RNA oligomers. Mutation of this “second gate” residue E664 disables the checkpoint and relieves the synthetic block and, together with mutation of the steric gate residue Y409, enables processive RNA synthesis in the double mutant polymerase TNQ, which is capable of synthesising the 65-nucleotide supF tRNA gene in <10 minutes (
Having established that mutation of E664 plays a key role in enabling processive RNA synthesis, we randomized position 664 in TN and in order to identify an optimal “second gate” residue. We screened for enhanced RNA polymerase activity using a novel high-throughput polymerase activity assay (PAA), based on capture of primer extension products and their quantification via hybridization to a specific antisense probe. The PAA screen identified several mutations promoting RNA polymerase activity (E664 K, L, Q, R), with lysine (E664K) proving most effective (
Following the success of this strategy we randomized position 409 in the context of the E664K mutation and performed an analogous PAA screen. This identified several promising mutations (Y409 A, G, P) of which G and P proved most advantageous (
We conclude that there are two key positions in the B-family DNA polymerase structure that inhibit RNA synthesis in the wild-type polymerase, and that mutation of both is essential for the engineering of a processive RNA polymerase. One, the steric gate residue (Y409), lies in the active site and prevents incorporation of NTPs by steric exclusion and is thus best mutated to G to provide maximum steric flexibility in the active site. The other, the “second gate” residue (E664), blocks the synthesis of longer RNA oligomers and is best mutated to K, reversing the electrostatic charge at this position.
Synthesis of Protein Coding and Functionalized mRNAs Using the Engineered RNA Polymerase TGK
Encouraged by the striking activity of TGK in tRNA synthesis, we challenged TGK to generate substantially longer RNAs. We first tested synthesis of the 748 nucleotide mRNA encoding green fluorescent protein (GFP). Astonishingly, TGK generated a full length GFP RNA in less than 10 minutes as judged by agarose gel-electrophoresis, RT-PCR and sequencing (
We determined the fidelity of RNA synthesis by TGK by a two-step RT-PCR protocol (below) using both forward synthesis and reverse transcription primers comprising a unique mismatch unambiguously identifying PCR products derived from RNA synthesis. Analysis of 8.2 kb of sequence data of Luciferase mRNA revealed a misincorporation rate of 5.5×10−4, which is comparable to the error rate of T7 RNA polymerase (2.1×10−4) and to the fidelity of TgoT with dNTPs (7.5×10−3,
Although the effect of mutating the steric gate residue on RNA synthesis has been investigated in some detail and its mechanism has been rationalized using structural and biochemical data, the basis for the striking effect of the “second gate” mutation on processive RNA synthesis was unclear. We sought to determine critical parameters of second gate function, in an attempt to better understand its mechanism. To this end we examined not just TGK and the parent polymerase TgoT, but also the intermediates TYK (TgoT E664K) and TGE (TgoT Y409G) for their RNA polymerase activity and processivity with RNA, DNA and chimeric primers.
As shown, TGK can efficiently and processively extend both DNA and RNA primers with NTPs. In contrast, TgoT can not extend RNA primers (except under highly forcing conditions) and displays strong extension termination after the 6th NTP incorporation from DNA primers. There appears to be a fundamental block to extension beyond a stretch of six ribonucleotides. When using DNA-RNA chimeric primers comprising one (or more) 3′-terminal NMPs, there is again a strong termination once a stretch of 6 NMPs is reached (i.e., extension of a primer with 1 terminal NMP is stalled after incorporation of a further 5 NMPs and a primer with 6 terminal NMPs is not extended,
The second gate also appears to control other aspects of polymerase substrate specificity. Both TGK and TYK (but not TgoT and TGE) are capable of trans-lesion synthesis (TLS,
Thus, we demonstrate that the thumb subdomain of DNA polymerases makes many important interactions with the nascent DNA duplex. Swan et al., Nat. Str. Mol. Biol. 16:979 (2009); Wang et al., Biochem. 50:581 (2011). Mutations in this critical region for polymerase function can cause a loss in fidelity, processivity and DNA binding affinity and have been implicated in genomic instability and disease. Kirchner et al., Genet. 155:1623 (2000); Kokoska et al., Mol. Cell Biol. 20:7490 (2000); Stocki et al., J. Mol. Biol. 254:15 (1995); Kasiviswanathan et al., J. Biol. Chem. 284:19501 (2009); Loh & Loeb, DNA Repair 4:1390 (2005). Here we have discovered an important post-synthetic substrate specificity checkpoint within the thumb domain of a replicative polymerase. Its inactivation by mutation releases a block on the synthesis of non-cognate nucleic acids and bypass of template lesions. Together with mutation of the steric gate residue, this “second gate” mutation converts a DNA polymerase into a primer-dependent RNA polymerase capable of the synthesis of 100-mer RNAs in seconds and of protein coding mRNAs of up to 1.7 kb in minutes to hours with fidelity comparable to the industry standard T7 RNA polymerase.
The existence of a synthetic checkpoint located in the polymerase thumb domain, was predicted by us and others (Astatke et al., 1998a; Brown & Suo, 2011; Xia et al., 2002; Ong et al., 2006) on the basis of multiple lines of evidence including termination of extension after synthesis of short oligomers (typically n+6) and modelling of a nascent RNA-DNA heteroduplex (Ong et al., 2006). Indeed, eight of the nine mutations in D4 (P657T, E658Q, K659H, Y663H, E664Q, D669A, N671K, I676T) are located in a sequence segment (P657-T676) in the centre of the thumb domain and in close contact with the nascent strand around n+6. Using iterative cycles of screening and site-directed mutagenesis, we narrowed the critical functional determinant down to a single residue (E664), mutation of which proved both necessary and sufficient for processive RNA polymerase activity. This key residue, which we termed the “second gate,” is most effectively mutated to the positively charged lysine (E664K) but mutation to an uncharged glutamine (E664Q) already provides significant RNA polymerase activity. This trend suggests that the negative charge of the parental E664 positioned in proximity to the nascent strand may be an important component of its function to arrest RNA synthesis.
Steric gate function is based on steric exclusion of the 2′OH on the incoming nucleotide. Brown & Sou, 2011; Kirouac et al., 2011. The mechanistic basis of second gate function is currently not clear, however, and may involve mechanisms other than geometric control. Indeed, some of the improvement in RNA synthesis may be caused by a 3- to 10-fold increase in affinity for both DNA:DNA and DNA:RNA primer-template duplexes mediated by the second gate mutation, as determined by electrophoretic mobility shift assay (EMSA). Mutation of the second gate residue E664 removes a repulsive negative charge in the polymerase thumb domain close to the nascent strand phosphate backbone and this may increase affinity for the primer-template duplex. The striking termination at n+6 in polymerases lacking the E664 mutation (TgoT, TGE), however, strongly suggests a second, geometric component to the mechanism.
Formulation of a steric model is hindered by the fact that there are currently no structures of a tertiary complex of an archeal B-family polymerase. In the existing B-family polymerase structures the critical region in the polymerase thumb domain sequence is divergent from the archaeal sequence (Swan et al., 2009; Franklin et al., 2001; Wang & Yang, Cell 139:1279 (2009); Liu et al., J. Biol. Chem. 281:18193 (2006); Savino et al., Struct. 12:2001 (2004)), making comparisons by analogy problematic. We therefore used the structure of a secondary complex of a mutant of the closely related archaeal polB Pfu (S. Wynne, P H, A. Leslie, unpublished results) as well as the tertiary complex structure of more divergent RB69 DNA polymerase (Wang et al., 2011) to model a nascent RNA strand on a DNA template (based on the NMR structure of an RNA-DNA heteroduplex (PDB: 1EFS (Hantz et al., Int. J. Macromol. 28:273 (2001));
The poor geometric complementarity of the RNA-DNA heteroduplex with the thumb domain contrasts with a much better fit in the polymerase active site. Indeed, the structures of tertiary complexes of A- and X-family DNA polymerases show that the preferred sugar pucker in and close to the active site is C3′-endo and the helical conformation of the primer-template duplex (up to n+3) is A-like but undergoes a conformational switch to cognate B-DNA as the primer-template duplex interacts with the thumb domain and exits the polymerase. Kiefer et al., Nat. 391:304 (1998); Pelletier Sci. 264:1891 (1994); Li et al., EMBO J. 17:7514 (1998). This conformational switch has previously been observed to play a role in polymerase specificity, whereby direct structural observation of the incorporation and extension of a G·T mismatch revealed a disruption of the A- to B-form transition by the mismatch that was “transmitted” back to the active site through the template strand and promoted stalling of the polymerase. Jonson & Beese, Cell 166:803 (2004). Such post-synthetic crosstalk between the second gate checkpoint in the thumb domain and the polymerase active site may similarly mediate the stalling at n+6 observed in RNA synthesis. The second gate checkpoint may therefore detect an emergent feature of the nascent nucleic acid duplex, possibly a non-cognate helical conformation.
The fact that the most effective second gate mutation (E664K) results in a charge reversal rather than removing side-chain bulk suggests that E664 does not function as a simple steric obstruction per se. Indeed, the steric clash at n+6 in our model does not occur directly with residue E664. Rather E664 but may act as an electrostatic “rudder,” steering the nascent strand to allow readout of helical parameters by the thumb domain. Alternatively, mutation of E664 may lead to structural rearrangements of the thumb domain, rendering it more permissive to non B-form nucleic acids. In either case, mutation of the second gate residue would allow the polymerase thumb domain to accommodate a range of helical conformations. Thus, the relaxed substrate specificity and lesion bypass ability of TGK would arise from relaxation of the geometric control of the positioning of the nascent and template strands in the active site, preventing stalling of polymerase extension upon encounter of a non-cognate duplex conformation.
Although highly conserved among polB family members of the Thermococcales, the identity of the second gate residue 664 (or equivalent) varies among more distantly related members of the polB family. Yet structures of the tertiary complexes in both S. cerevisiae Pol delta and E. coli Pol II indicate at least a broad conservation of its structural neighbourhood in the thumb domain. It therefore seems possible that similar “second gate” checkpoints exist in the wider polB family (and indeed in other families) but, in analogy to the steric gate residue which varies among polymerase families (
Exclusion of RNA from the genome is important for genome stability in mesophilic organisms like S. cerevisiae (Nick McElhinny et al., Nat. Chem. Biol. 6:774 (2010)) and might be of even higher significance for thermophiles. However, the striking effect of the second gate mutation on TLS suggest that it may have biological functions that go beyond the prevention of RNA synthesis. These may include the post-synthetic recognition of misincorporation and/or read-through of DNA damage mirroring the read-ahead recognition of template uracil and hypoxanthin in the same class of archeal polymerases. Firbank et al., J. Mol. Biol. 381:529 (2008).
TGK differs in several potentially useful ways from the single-subunit RNAPs like T7 RNA polymerase, including thermostability and, most importantly, the ability to initiate RNA synthesis from an oligonucleotide primer. The engineered polymerase TGK is thus the first thermostable primer-dependent processive RNA polymerase. Primer-dependent RNA synthesis from a DNA template is a phenotype not commonly observed in nature, where RNA synthesis is typically initiated de novo from a specific promoter sequence (e.g., T7 RNA polymerase) (Steitz, Curr. Opin. Str. Biol. 14:4 (2004)), or a protein transcription factor complex, although there are examples where RNA synthesis is initiated from a short RNA primer derived by “Cap-snatching” from cellular mRNAs (e.g., viral RNA-dependent RNA polymerases) (Plotch et al., Cell 23:847 (1981); Mir et al., PNAS 105:19294 (2998)). Although T7 RNA polymerase is capable of primer dependent RNA synthesis (Rusakova et al., FEBS Lett. 423:189 (1998); Ivanov et al., Mol. Biol. 38:798 (2004)) it is weak and non-processive, presumably because binding to a pre-annealed DNA primer does not trigger the conformational change to the processive form of T7 RNA polymerase (Yin et al., Sci. 298:1387 (2002)).
Primer-dependent RNA synthesis is potentially useful for a number of applications as it allows free choice of the RNA 5′ end or RNA 5′ UTR chemistry. TGK is capable of extending a wide variety of primers including those bearing 5′ groups such as Biotin, Cy3 or FITC as well as both DNA and RNA primer oligonucleotides comprising internal alphaS, 2′OMe or LNA modifications. Indeed, we have yet to identify a primer modification which reduces extension by TGK to a significant degree. Furthermore, TGK frees enzymatic RNA synthesis from the T7 RNA polymerase need to initialise transcription with a G.
TGK also displays an expanded substrate spectrum with respect to the incorporation of modified NTPs. For example, TGK allowed the synthesis of the 1.7 kb Luciferase mRNA fully substituted with 5-methyl-C and pseudouridine (Ψ), with potential applications in gene therapy and stem cell reprogramming as shown recently. Warren et al., Cell Stem Cell (2010); Kormann et al., Nat. Biotechnol. 29:154 (2011). Furthermore, TGK efficiently synthesises fully 2′-azido (2′-N3) or 2′-fluoro (2′-F) substituted DNA with applications in the evolution of nuclease resistant aptamers with expanded chemistries. Watts et al., Org. Biol. Chem. 7:1904 (2009); Ono et al., Nucl. Acids Res. 25:4581 (1997). Although overnight synthesis of 2′F-RNA had been described previously (Ono et al., 1997), TGK is capable of synthesis of fully substituted 2′F-RNAs in minutes.
In conclusion, our results suggest that the adaptive path from DNA polymerase to RNA polymerase is surprisingly short. Although our “wild-type” polymerase TgoT contained four mutations, disabling exonuclease (D141A, E143A) and uracil stalling (V93Q) functions as well as the Therminator mutation (A485L) to increase substrate promiscuity, these did not confer more than baseline RNA polymerase activity. Thus the “evolutionary jump” to RNA polymerase activity involved mutation of just two residues: the classic steric gate (Y409) in the active site and the newly described “second gate” (E664) in the thumb domain, which together enabled not only processive RNA polymerase activity but TLS and synthesis of highly modified RNA polymers. The identification of a “second gate” in the polymerase thumb subdomain indicates that a key determinant of polymerase substrate specificity is located outside the active site and exerts its influence after the initial incorporation and extension steps.
Detailed Description of the Drawings for Example 24(a) Structure of TGK, mapped onto the homologous E. coli DNA pol II (3MAQ). Ono et al., 1997. The key mutations forming the 2-step adaptive path are shown. Other mutations from wild type (V93Q, D141A, E143A, A485L) are shown, as are the template strand and the primer strand. The incoming dGTP is shown. (b) RNA:DNA hybrid duplex (1EFS) (Wang et al., 2011) modelled into the ternary complex of Pfu (S. Wynne and A. Leslie, unpublished structure) showing the clash generated when nucleic acid intermediate between A- and B-form is synthesised by a DNA polymerase. (c) As (b), except with the E664K mutation showing the charge reversal.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described aspects and embodiments of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the following claims.
Claims
1. A recombinant nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a DNA nucleotide polymer template, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated, relative to the amino acid sequence of SEQ ID NO:1, at one or more residues of the thumb region, said residues selected from amino acids 651 to 679 (patch 10A); and wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at residue E664.
2. The recombinant nucleic acid polymerase of claim 1, wherein said non-DNA nucleotide polymer is a RNA polymer; and wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at residue Y409.
3. The recombinant nucleic acid polymerase of claim 2, wherein said polymerase comprises the mutations Y409G and E664K.
4. A method for making a RNA nucleotide polymer, said method comprising contacting a DNA template with a nucleic acid polymerase of claim 2 and incubating to allow polymerization.
5. A method for making a RNA nucleotide polymer, said method comprising contacting a DNA template with a nucleic acid polymerase of claim 3 and incubating to allow polymerization.
6. The recombinant nucleic acid polymerase of claim 1, wherein said polymerase is capable of producing a HNA nucleotide polymer from a DNA nucleotide polymer template and said polymerase comprises amino acid sequence corresponding to amino acids 651 to 679 (patch 10A) of SEQ ID NO:12.
7. The recombinant nucleic acid polymerase of claim 6, wherein said polymerase is capable of producing a HNA nucleotide polymer from a DNA nucleotide polymer template and said polymerase comprises amino acid sequence corresponding to SEQ ID NO:12.
8. A recombinant nucleic acid polymerase capable of reverse transcribing a HNA nucleotide polymer into a DNA nucleotide polymer, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated, relative to the amino acid sequence of SEQ ID NO:1, at residue I521.
9. The recombinant nucleic acid polymerase according to claim 8, wherein said polymerase comprises a mutation selected from the group consisting of I521L, I521P and I521H.
10. The recombinant nucleic acid polymerase of claim 9, wherein said polymerase comprises the mutation I521L.
11. The recombinant nucleic acid polymerase of claim 8, wherein said polymerase further comprises the mutation A485L.
12. The recombinant nucleic acid polymerase of claim 11, wherein said polymerase further comprises the mutation V93Q.
13. The recombinant nucleic acid polymerase of claim 12, wherein said polymerase further comprises the mutations E141A and E143A.
14. The recombinant nucleic acid polymerase of claim 8, wherein said non-DNA polymer is a HNA polymer and wherein said polymerase comprises the mutations I521L, A485L, V93Q, E141A, and E143A.
15. A system comprising:
- (a) a recombinant nucleic acid polymerase capable of producing a non-DNA nucleotide polymer from a DNA nucleotide polymer template, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated, relative to the amino acid sequence of SEQ ID NO:1, at one or more residues of the thumb region, said residues selected from amino acids 651 to 679 (patch 10A); and wherein said amino acid sequence is mutated relative to the amino acid sequence of SEQ ID NO:1 at residue E664; and
- (b) a recombinant nucleic acid polymerase capable of reverse transcribing a HNA nucleotide polymer into a DNA nucleotide polymer, said polymerase comprising amino acid sequence having at least 36% identity to the amino acid sequence of SEQ ID NO:1, wherein said amino acid sequence is mutated, relative to the amino acid sequence of SEQ ID NO:1, at residue I521.
Type: Application
Filed: Mar 13, 2018
Publication Date: Aug 23, 2018
Applicant: MEDICAL RESEARCH COUNCIL (Swindon)
Inventors: Philipp Holliger (Cambridge), Christopher Cozens (Cambridge), Vitor B. Pinheiro (Cambridge), Alexander I. Taylor (Cambridge)
Application Number: 15/920,345
