MODIFIED POLYMERASES FOR REPLICATION OF THREOSE NUCLEIC ACIDS

• US 20160097069A1
• Filed: 08/06/2015
• Published: 04/07/2016
• Est. Priority Date: 08/19/2014
• Status: Active Grant

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Chaput, John, Dunn, Matthew
• Granted Patent: US 9,469,862 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12P 7/34 produced from substrate containing protein as nitrogen source
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

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 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 amino acid sequence comprises the amino acid sequence of SEQ ID NO:

1.

4. The nucleic acid of claim 1, wherein the amino acid sequence consists of SEQ ID NO:

1.

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

2.

6. The nucleic acid of claim 5, wherein the nucleotide sequence of the nucleic acid consists of SEQ ID NO:

2.

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

8. A recombinant cell comprising the nucleic acid expression vector of claim 7.

9. The recombinant cell of claim 8, wherein the recombinant cell is a prokaryotic cell.

14. A kit comprising the purified TNA polymerase of claim 1 and at least one threose nucleotide.

15. The kit of claim 14, wherein the at least one threose nucleotide comprises tA, tT, tG, and tC.

10. A purified 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.

11. The TNA polymerase of claim 10, wherein the amino acid sequence is at least 98% identical to SEQ ID NO:

1.

12. The TNA polymerase of claim 10, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

1.

13. The TNA polymerase of claim 10, wherein the amino acid sequence of the TNA polymerase consists of SEQ ID NO:

1.

16. A method for synthesizing a TNA, comprising contacting a DNA template with:

(i) a 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; and

(ii) threose nucleotides under conditions that permit TNA polymerization.

17. The method of claim 16, wherein the amino acid sequence is at least 98% identical to SEQ ID NO:

1.

18. The method of claim 16, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

1.

19. The method of claim 16, wherein the amino acid sequence consists of SEQ ID NO:

1.