METHDS AND COMPOSITIONS FOR REPLICATION OF THREOSE NUCLEIC ACIDS

• US 20160138063A1
• Filed: 01/02/2014
• Published: 05/19/2016
• Est. Priority Date: 01/04/2013
• Status: Abandoned Application

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Yu, Hanyang, Zhang, Su, Dunn, Matthew R., Chaput, John
• Application Number:
  US14/758,844
• Publication Number:
  US 20160138063A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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 method for synthesizing a threose nucleic acid polymer, comprising:

contacting a single stranded DNA template hybridized to a primer with a DNA polymerase comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO;

1 in the presence of tTTP, tGTP, tATP; and

(i) dCTP;

or (ii) a combination of tCTP and dCTP; and

incubating at a temperature suitable for polymerization by the DNA polymerase to obtain a threose nucleic acid.

2. The method of claim 1, wherein the DNA polymerase is in the presence of tTTP, tGTP, tATP, and dCTP.

3. The method of claim 2, wherein the contacting step is done in the substantial absence of tCTP.

4. A threose nucleic acid generated by the method of claim 3

5. The method of claim 1, wherein the DNA polymerase is in the presence of tATP, tTTP, tGTP, and a combination of tCTP and dCTP.

6. The method of claim 1, wherein the DNA polymerase comprises the amino acid sequence of SEQ ID NO:

1.

7. The method of claim 1, wherein the single stranded DNA template sequence is restricted to the nucleotides dA, dC, and dT.

8. The method of claim 1, wherein the single stranded DNA template sequence comprises 7-deaza-dGTP instead of dGTP.

9. A method for reverse transcribing a threose nucleic acid, comprising contacting a threose nucleic acid template with a SuperScript II reverse transcriptase in the presence of a primer and dNTPs, dNTP analogs, or a combination thereof to obtain a threose nucleic acid reverse-transcription mix, and incubating the mix at a temperature suitable for SuperScript II reverse transcriptase activity to obtain a cDNA copy of the threose nucleic acid template, wherein the threose nucleic acid template comprises deoxycytidine.

10. A method for molecular evolution of threose nucleic acids, the method comprising:

(i) providing a DNA template library comprising 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% identical to the amino acid sequence of SEQ ID NO;

1 in the presence of tTTP, tGTP, tATP; and

(i) dCTP;

or (ii) a combination of tCTP and dCTP; and

incubating at 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 to obtain a selected DNA template library.

11. The method of claim 10, wherein the diverse DNA template sequences are restricted to the nucleotides dA, dC, and dT.

12. The method of claim 10, wherein the selection assay in step (iv) comprises selection of one or more cTNAs based on affinity for a ligand.

13. The method of claim 10, wherein the selection assay in step (iv) comprises selection of one or more cTNAs based on a catalytic activity.

14. The method of claim 10, wherein the selection assay in step (iv) comprises selection of one or more cTNAs based on fluorescence emission.

15. The method of claim 10, wherein step (iii) is done in the substantial absence of tCTP.

16. The method of claim 10, wherein the DNA template library comprises DNA templates comprising 7-deaza-dGTP instead of dGTP.

17. A TNA transcription system comprising a single stranded DNA template, a DNA polymerase comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

1, tTTP, tGTP, tATP; and

(i) dCTP;

or (ii) a combination of tCTP and dCTP.

18. The TNA transcription system of claim 17, wherein the TNA transcription system comprises dCTP, but is substantially free of tCTP.

19. The TNA transcription system of claim 17, wherein the single stranded DNA template comprises 7-deaza-dGTP instead of dGTP.

20. A TNA reverse transcription system comprising a TNA template, a SuperScript II reverse transcriptase, and dNTPs;

wherein the TNA template comprises dC.