Nucleoside triphosphate transporter and uses thereof

Patent metadata

Field	Value
Publication number	US-10696720-B2
Application number	US-201916546100-A
Country	US
Kind code	B2
Filing date	Aug 20, 2019
Priority date	Jun 24, 2016
Publication date	Jun 30, 2020
Grant date	Jun 30, 2020

Abstract

Disclosed herein are proteins, methods, cells, engineered microorganisms, and kits for generating a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum . Also disclosed herein proteins, methods, cells, engineered microorganisms, and kits for production of a nucleic acid molecule that comprises an unnatural nucleotide utilizing a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum.

First claim

What is claimed is:

1. A method of increasing production of a nucleic acid molecule in an E. coli cell, comprising:
(a) expressing in the cell a recombinant nucleoside triphosphate transporter, wherein the recombinant nucleoside triphosphate transporter has an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8,
(b) incubating the cell with one or more unnatural nucleoside triphosphates, and
(c) transporting the one or more unnatural nucleoside triphosphates into the cell using the recombinant nucleoside triphosphate transporter, wherein the nucleic acid molecule comprises one or more unnatural nucleotides comprising a base selected from the group consisting of

2. The method of claim 1 , wherein the nucleic acid molecule comprises DNA.

3. The method of claim 1 , wherein the nucleic acid molecule comprises RNA.

4. The method of claim 1 , wherein the nucleic acid molecule comprises one or more unnatural nucleotides comprising a base which is

5. The method of claim 1 , wherein the nucleic acid molecule comprises one or more unnatural nucleotides comprising a base which is

6. The method of claim 1 , wherein the one or more unnatural nucleoside triphosphates comprises a base selected from the group consisting of

7. The method of claim 1 , wherein the one or more unnatural nucleoside triphosphates comprises a base which is

8. The method of claim 1 , wherein the one or more unnatural nucleoside triphosphates comprises a base which is

9. A method of increasing production of a DNA nucleic acid molecule in an E. coli cell comprising:
(a) expressing in the cell a recombinant nucleoside triphosphate transporter, wherein the recombinant nucleoside triphosphate transporter has an amino acid sequence comprising selected from SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8,
(b) incubating the cell with one or more unnatural 2′-deoxyribonucleoside triphosphates, and
(c) transporting the one or more unnatural 2′-deoxyribonucleoside triphosphates into the cell using the recombinant nucleoside triphosphate transporter, wherein the one or more unnatural 2′-deoxyribonucleoside triphosphates comprise a base selected from the group consisting of:

10. A method of increasing production of a RNA nucleic acid molecule in an E. coli cell, comprising:
(a) expressing in the cell a recombinant nucleoside triphosphate transporter, wherein the recombinant nucleoside triphosphate transporter has an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8,
(b) incubating the cell with one or more unnatural ribonucleoside triphosphates, and
(c) transporting the one or more unnatural ribonucleoside triphosphates into the cell using the recombinant nucleoside triphosphate transporter, wherein the one or more unnatural ribonucleoside triphosphates comprise a base selected from the group consisting of:

11. The method of claim 1 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 4.

12. The method of claim 11 , wherein the one or more unnatural nucleoside triphosphates comprises a base selected from

13. The method of claim 1 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 6.

14. The method of claim 13 , wherein the one or more unnatural nucleoside triphosphates comprises a base selected from

15. The method of claim 1 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 8.

16. The method of claim 15 , wherein the one or more unnatural nucleoside triphosphates comprises a base selected from

17. The method of claim 9 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 4.

18. The method of claim 17 , wherein the one or more unnatural 2′-deoxyribonucleoside triphosphates comprises a base selected from

19. The method of claim 9 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 6.

20. The method of claim 19 , wherein the one or more unnatural 2′-deoxyribonucleoside triphosphates comprises a base selected from

21. The method of claim 9 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 8.

22. The method of claim 21 , wherein the one or more unnatural 2′-deoxyribonucleoside triphosphates comprises a base selected from

23. The method of claim 10 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 4.

24. The method of claim 23 , wherein the one or more unnatural ribonucleoside triphosphates comprises a base selected from

25. The method of claim 10 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 6.

26. The method of claim 25 , wherein the one or more unnatural ribonucleoside triphosphates comprises a base selected from

27. The method of claim 10 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 8.

28. The method of claim 27 , wherein the one or more unnatural ribonucleoside triphosphates comprises a base selected from

Assignees

• Scripps Research Inst

Inventors

• Romesberg Floyd E
• Zhang Yorke

Classifications

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title