Modified polymerases for replication of threose nucleic acids

Abstract

Methods and compositions for replication of threose nucleic acids (TNAs) are described. The described methods include a method for transcribing a DNA template into a TNA, and a method for reverse transcribing a threose nucleic acid into a cDNA.

Claims

1. A nucleic acid encoding a threose-nucleic acid (TNA) polymerase comprising an amino acid sequence at least 95% identical to SEQ ID NO:1, wherein the residue corresponding to 141 of SEQ ID NO:1 is an alanine, residue 143 is an alanine, residue 485 is an arginine, and residue 664 is an isoleucine; and wherein the encoded DNA polymerase synthesizes a TNA in the presence of a DNA template and threose nucleotides.

2. The nucleic acid of claim 1, wherein the amino acid sequence is at least 98% identical to SEQ ID NO:1.

3. The nucleic acid of claim 1, wherein the nucleotide sequence of the nucleic acid comprises SEQ ID NO:2.

4. The nucleic acid of claim 3, wherein the nucleotide sequence of the nucleic acid consists of SEQ ID NO:2.

5. A nucleic acid expression vector comprising the nucleic acid of claim 1.

6. A recombinant cell comprising the nucleic acid expression vector of claim 5.

7. The recombinant cell of claim 6, wherein the recombinant cell is a prokaryotic cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

(2) FIG. 1. DNA and protein sequence of 9N-RI polymerase. 9N-RI contains the following amino acid substitutions with respect to 9N: D141A, E143A, A485R, and E664I.

(3) FIG. 2A. TNA transcription by 9N-RI polymerase of G series templates in the presence and absence of manganese ion. Diagram of the TNA transcription reaction depicting the four different sequences with increasing number of G nucleobases.

(4) FIG. 2B. TNA transcription by 9N-RI polymerase of G series templates in the presence and absence of manganese ion. 9N-RI polymerase produces full-length TNA molecules in both the presence and absence of manganese ions. 9N-RI is the first polymerase capable of producing large quantities of TNA in the absence of manganese ions.

(5) FIG. 3. Fidelity of 9N-RI in the absence of manganese. RI has a fidelity of 99.36% after sequencing 2350 nucleobases.

(6) FIG. 4. 9N-RI polymerase is capable of generating pure TNA products as well as mixed TNA-DNA molecules. The gel on the left depicts the production of pure TNA molecules over time from 5 to 50 hours. The gel on the right depicts chimeric TNA-DNA molecules composed of tA, tG, tT, and dC.

DETAILED DESCRIPTION

(7) Disclosed herein is are methods, compositions and systems for replication and in vitro evolution of TNAs based on the unexpected finding that certain TNA synthesis conditions, as described herein, permit the efficient and faithful synthesis of XNAs from DNA templates and their reverse transcription into cDNAs using known polymerases.

(8) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

(9) In describing the embodiments and claiming the invention, the following terminology will be used in accordance with the definitions set out below.

(10) As used herein, about means within 5% of a stated range within the relevant parameter.

(11) As used herein, TNA or TNAs refer to nucleic acids having a backbone composed primarily of -L-threofuranosyl-(3.fwdarw.2) (threose)-containing nucleotides, but may include heteropolymers comprising both tNTPs and dNTPs (e.g., dC).

(12) As used herein, TNTPs refer to threose nucleotide triphosphates.

(13) As used herein, TNTP analog refers to a threose nucleotide triphosphate having a modified base moiety.

(14) As used herein, TNA polymerase refers to a polymerase capable of utilizing a DNA template and tNTPs to synthesize a complementary TNA sequence.

(15) With respect to the amino acid sequence homology of polypeptides described herein, one of ordinary skill in the art will appreciate that structural and functional homology of two or polypeptides generally includes determining the percent identity of their amino acid sequences to each other. Sequence identity between two or more amino acid sequences is determined by conventional methods. See, for example, Altschul et al., (1997), Nucleic Acids Research, 25(17):3389-3402; and Henikoff and Henikoff (1982), Proc. Natl. Acad. Sci. USA, 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the BLOSUM62 scoring matrix of Henikoff and Henikoff (ibid.). The percent identity is then calculated as: ([Total number of identical matches]/[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).

(16) Described herein are TNA polymerases, nucleic acids encoding such TNA polymerases, and methods for synthesizing TNAs using DNA as a template. In various embodiments the TNA polymerase comprises an amino acid sequence at least 95% (e.g., 97%, 98%, 99%, or 100%) identical to the amino acid sequence of 9N-RI polymerase shown below as SEQ ID NO:1.

(17) SEQ ID NO:1; amino acid sequence of Therminator DNA polymerase.

(18) TABLE-US-00001 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIED VKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYENHPQDVPAIRDRI RAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFAIATLYHEGE EFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKRFLRVVRE KDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAV EVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWE SGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTG NLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGGYVKEPERGLWDNI VYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIP SLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRLIKILANSFYGYYGYA KARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPG ADAETVKKKAKEFLKYINPKLPGLLELEYEGFYVRGFFVTKKKYAVIDEE GKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKL SKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVIS YIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGY RKEDLRYQKTKQVGLGAWLKVKGKK.

(19) In some embodiments, the DNA polymerase comprises an A485L point mutation relative to the amino acid sequence of the 9N DNA polymerase and is greater than about 95% identical to the amino acid sequence of Therminator DNA polymerase (Therminator DNA polymerase), e.g., about 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of Therminator DNA polymerase. In one embodiment, the DNA polymerase to be used comprises the amino acid sequence of SEQ ID NO:1. Typically, TNA synthesis using the Therminator polymerase is carried out at about 50 C. to about 60 C. In some embodiments, the TNA synthesis reaction is carried out at about 55 C.

(20) Suitable concentrations of tNTPs range from about 100 M to about 1000 M, e.g., about 25, 30, 35, 40, 50, 60, 70, 80, or another concentration of tNTPs from about 100 M to about 1000 M.

(21) In some embodiments, the single stranded DNA template to be used in the method comprises a sequence that is restricted to the nucleotides dA, dC, and dT. While not wishing to be bound by theory, it is believed that by limiting single stranded templates to sequences containing these three nucleotides, the fidelity of the sequence transcribed into TNAs is significantly increased as described herein. Also encompassed herein are heteropolymeric TNAs generated by the above-described method, which include tA, tT, tG, and dC.

(22) The fidelity is greatly increased in the presence of Mn if you don't have dG in the templates. These templates transcribe with high efficiency very quickly. Adding the fourth nucleotide to the react greatly reduces the fidelity in the presence of Mn while also reducing its efficiency.

(23) Also described herein is method for reverse transcribing a TNA. In various embodiments, a TNA is reverse transcribed by a method that includes: contacting a TNA template that contains dCTP with a SuperScript II reverse transcriptase in the presence of a primer and dNTPs, and incubating the resulting mix, at a temperature suitable for SuperScript II reverse transcriptase activity, to obtain a cDNA copy of the TNA template. We can also reverse transcribe sequences containing tCTP in the TNA strand. Typically the reverse transcription reaction using the SuperScript II reverse transcriptase is carried out at a temperature of about 37 C. to about 45 C. In some embodiments, the TNA reverse transcription reaction is carried out at 42 C. Also disclosed herein is a method for molecular evolution of threose nucleic acids, which includes the steps of: (i) providing a DNA template library containing diverse DNA template sequences; (ii) hybridizing the template library with one or more complementary primer sequences; (iii) incubating the hybridized template library with a DNA polymerase comprising an amino acid sequence at least 95% (e.g., 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1 in the presence of tTTP, tGTP, tATP, and dCTP, and incubating at a temperature suitable for polymerization by the DNA polymerase to obtain a cTNA library; (iv) subjecting the cTNA library to a selection assay to obtain at least one or more selected cTNAs; and (v) incubating the one or more selected cTNAs with a primer, a SuperScript II reverse transcriptase, and dNTPs at a temperature suitable for SuperScript II reverse transcriptase activity to obtain a selected DNA template library. In some embodiments, the diverse DNA template sequences are restricted to dA, dC, and dT, although the enzyme RI can do all four nucleotides.

(24) TNAs can be selected from a cTNA library in step (iv) based on a number of different criteria and assays depending on a desired functionality or endpoint for the TNAs being generated. Accordingly, in some embodiments the selection assay in sep (iv) includes selection of one or more cTNAs from the cTNA library based on affinity for a ligand. Examples of suitable affinity assays known in the art include, but are not limited to, aptamer affinity chromatography, systematic evolution of ligands by exponential enrichment (SELEX), and kinetic capillary electrophoresis. In other embodiments, selection of one or more cTNAs from the cTNA library is based on a catalytic activity. Methods for assaying and selecting catalytic activities, e.g., ribozyme activities, are known in the art as described in, e.g., Link et al. (2007), Biol Chem 388(8):779-786. In some embodiments, one or more cTNAs are selected based on a desired fluorescence emission. See, e.g., Paige et at (2011), Science, 333(6042):642-646.

(25) In the various methods described herein, hybridization between a primer and its target sequence is generally carried out under high stringency conditions under which the primer is annealed with its complementary template sequence at a temperature approximately 5 C. below the primer's melting temperature T.sub.m.

(26) Also described herein are TNA transcription systems. In various embodiments a TNA transcription system includes the following components: a single stranded DNA template, a DNA polymerase comprising an amino acid sequence at least 95% identical to the amino acid sequence of Therminator DNA polymerase, tTTP, tGTP, tATP; and (i) dCTP; or (ii) a combination of tCTP and dCTP.

(27) Also disclosed herein are TNA reverse transcription systems. Generally a TNA reverse transcription system, as described herein, includes: a TNA template comprising dC, a SuperScript II reverse transcriptase, and dNTPs.

(28) 9NRI is a more active TNA polymerase than Therminator polymerase and can be used for all of the same applications as Therminator DNA polymerase. 9NRI is better than Therminator in its ability to transcribe templates of increased complexity with higher efficiency. As a result, researchers can utilize 9NRI to generation more complex molecules with increasingly more sophisticated functions. 9NRI has potential to generate four nucleotide TNA molecules for in vitro selection of complex TNA molecules that are capable of performing complex functions. Additionally, since biologically relevant molecules are usually comprised of four nucleotides, the ability to generate four nucleotide TNA molecules provides potential targeting mechanism for silencing technology. Additionally, there has been much interest in the information storage capabilities of nucleic acids. Since TNA is inherently nuclease resistant, TNA has the potential to become a highly stable and long lasting medium for storing large amounts of information.

EXAMPLES

(29) The invention will be more fully understood upon consideration of the following non-limiting Examples. The invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.

Example 1

Generation of a Mutated DNA Polymerase for TNA Synthesis

(30) In this example, we show how to prepare the mutated DNA polymerase for TNA synthesis. FIG. 1 depicts the nucleotide and amino acid sequence of 9NRI polymerase. DNA template and its corresponding primer are heated in Thermopol buffer for 5 min at 95 C. The reaction is cooled for 10 min at 4 C to promote the formation of primer-template complexes. After the 10 minute incubation, the enzyme and manganese are combined and added to the primer template complex. The reaction is then brought to 55 C. The reaction is initiated by adding tNTPs to a final concentration of 100 uM each and then incubated for the desired amount of time.

Example 2

Identification of 9NRI

(31) In this example, we describe how we identified and characterized 9NRI and its mutations D141A, E143A, A485R, and E664I. FIG. 2 depicts TNA transcription reactions of 9NRI polymerase in the presence and absence of manganese. Four different templates containing an increase number of sequential G nucleobases were combined with radiolabeled primer, tNTPs, and enzyme in the presence or absence of manganese ions. The reactions were incubated for three hours when in presence of Mn2+ and 25 hrs when in the absence of Mn2+ at 55 C. The TNA transcription reactions were analyzed by denaturing urea polyacrylamide gel electrophoresis. The gels were then imaged on a phosphorimaging screen.

(32) Variants were generated for all 20 amino acids at positions 485 and 664 independently by site directed mutagenesis. Each variant was tested in a TNA transcription reaction and analyzed by polyacrylamide gel electrophoresis. The most active variants at positions 485 and 664 were then generated as double mutants to test for synergistic effects. The double mutants were screened in the exact method as the single mutants.

Example 3

Characterization of 9NRI

(33) In this example, we describe the characterization of the 9NRI enzyme. 9NRI is capable of generating a significant amount of full length TNA in the absence of manganese. Previous enzymes were unable to generate any material. Additionally, 9NRI is capable of transcribing through sequences with higher numbers of sequential G nucleobases.

Example 4

Functions of 9NRI

(34) In this example, we describe how 9NRI can synthesize a four nucleotide alphabet (A, T, C, and G) in the absence of manganese ions and why this is beneficial. 9NRI is capable of generating complex four nucleotide TNA polymers both in the presence and absence of manganese ions. Removing manganese from the transcription reactions greatly improves the fidelity of TNA transcription. The ability to generate four nucleotide TNA sequences enables the selection of biologically-relevant TNA molecules. Four nucleotide sequences have the potential to fold in more complex tertiary structures with more sophisticated functions. Additionally, since most biological genetic materials are composed of four nucleotides, four nucleotide TNA sequences can be generated to target those biomolecules. For example, nucleic acid pharmaceuticals. TNA's inherent nuclease resistance gives it even more promise for biopharmaceutical development.

Example 5

Fidelity of 9NRI

(35) In this example, we describe how we determined that 9NRI functions with high fidelity as a DNA dependent TNA polymerase. 9NRI was discovered by screening all 20 amino acids at positions 485 and 664. Variants were expressed in XL1-blue competent cells. Clarified cell lysate was used in primer extension reactions in the presence and absence of Manganese ions. Extension efficiency was determined by comparing TNA transcription products by polyacrylamide gel electrophoresis. Single variants with the highest activity were then to look for variant combinations that synergistically improved TNA transcription.

(36) FIG. 3 illustrates the fidelity of TNA transcription in the absence of Mn2+. To determine the fidelity, a DNA template of a fixed sequence was transcribed into TNA using 9NRI polymerase. TNA molecules were then separated from their DNA templates by polyacrylamide gel electrophoresis, cut out of the gel, electroeluted, and then concentrated by ethanol precipitation. Purified TNA molecules were then reverse transcribed into DNA and amplified by PCR. cDNA molecules were then cloned into pJET and analyzed by DNA sequencing. The chart demonstrates that TNA transcription in the absence of Mn2+ proceeds with very high fidelity as compared to in the presence of Mn2+. Removing the Mn2+ results in approximately 12 fold reduction in the error rate of TNA transcription.

Example 6

Efficiency of 9NRI

(37) In this example, we describe how we determined that 9NRI can synthesize oligonucleotides that have a DNA-TNA mixed sequence backbone with high efficiency and high fidelity.

(38) FIG. 4 demonstrates the generation of mixed backbone DNA-TNA chimeric molecules. The substitution of dCTP for tCTP during TNA transcription not only allows for the generation of chimeric molecules, but it also increases the efficiency of TNA transcription. These reactions were performed as in FIG. 2 in the absence of Mn2+. Reactions were sampled every 5 hours and each sampling was analyzed by PAGE and imaged by phosphorimaging.

(39) 9NRI was tested for TNA transcription activity with both tCTP and dCTP in the TNA transcription reactions. Products were analyzed by polyacrylamide gel electrophoresis. TNA transcription was more efficient and had a higher fidelity when dCTP was present.

Example 7

Use of 9NRI

(40) In this example, we describe how one of skill would use 9NRI. 9NRI is a highly faithful, highly efficient TNA polymerase. This polymerase enables to synthesis of complex, four-nucleotide TNA polymers. In one embodiment, these polymers can be used to generate libraries of TNA molecules for in vitro selection as well as for generating molecules capable of interacting with naturally occurring biomolecules. Other uses known to one of skill in the art can also apply.

(41) TABLE-US-00002 TABLE1 SequencesofTemplates Name Sequence 4NT.0G 5-ACTATTCAACTTACAATCCTATCAACCTTATAATCCAC (SEQID TTGGCTACTGCATACGAGTGTC-3 NO:3) 4NT.1G 5-ACTATTCAACTTACAATCGTATCAACCTTATAATCCAC (SEQID TTGGCTACTGCATACGAGTGTC-3 NO:4) 4NT.2G 5-ACTATTCAACTTACAATGGTATCAACCTTATAATCCAC (SEQID TTGGCTACTGCATACGAGTGTC-3 NO:5) 4NT.3G 5-ACTATTCAACTTACAATGGGATCAACCTTATAATCCAC (SEQID TTGGCTACTGCATACGAGTGTC-3 NO:6) PBS2 5-GACACTCGTATGCAGTAGCC-3 (SEQID NO:7)

(42) The invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.