Nucleoside triphosphate transporter and uses thereof

Patent metadata

Field	Value
Publication number	US-10696719-B2
Application number	US-201916546097-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 recombinant nucleoside triphosphate transporter having an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

2. The recombinant nucleoside triphosphate transporter of claim 1 , wherein the recombinant nucleoside triphosphate transporter is encoded by an extrachromosomal nucleic acid molecule.

3. The recombinant nucleoside triphosphate transporter of claim 2 , wherein the extrachromosomal nucleic acid molecule comprises a nucleic acid sequence provided in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

4. A prokaryotic cell comprising the recombinant nucleoside triphosphate transporter of claim 1 .

5. The prokaryotic cell of claim 4 , wherein the prokaryotic cell is an engineered cell.

6. The prokaryotic cell of claim 4 , wherein the prokaryotic cell is E. coli.

7. The prokaryotic cell of claim 4 , further comprising an unnatural nucleoside triphosphate transported into the prokaryotic cell using the recombinant nucleoside triphosphate transporter, wherein the unnatural nucleoside triphosphate comprises a base selected from the group consisting of:

8. The prokaryotic cell of claim 7 , wherein the unnatural nucleoside comprises a deoxyribose moiety.

9. The prokaryotic cell of claim 7 , wherein the unnatural nucleoside comprises a ribose moiety.

10. The prokaryotic cell of claim 4 , further comprising an unnatural base pair (UBP) comprising a first unnatural nucleoside and a second unnatural nucleoside, wherein the first unnatural nucleoside comprises a base that is

and the second unnatural nucleoside comprises a base selected from the group consisting of

11. The prokaryotic cell of claim 10 , wherein the second unnatural nucleoside comprises a base that is

12. The prokaryotic cell of claim 4 , wherein the prokaryotic cell is isolated and purified.

13. A nucleic acid molecule encoding the recombinant nucleoside triphosphate transporter of claim 1 .

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

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

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

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

18. The prokaryotic cell of claim 4 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 6.

19. The prokaryotic cell of claim 4 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 8.

20. The prokaryotic cell of claim 6 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 4.

21. The prokaryotic cell of claim 6 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 6.

22. The prokaryotic cell of claim 6 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 8.

23. The prokaryotic cell of claim 10 , wherein the cell is E. coli.

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

25. The prokaryotic cell of claim 23 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 6.

26. The prokaryotic cell of claim 23 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 8.

27. The prokaryotic cell of claim 11 , wherein the cell is E. coli.

28. The prokaryotic cell of claim 27 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 4.

29. The prokaryotic cell of claim 27 , wherein the recombinant nucleoside triphosphate transporter has amino acid sequence SEQ ID NO: 6.

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

Assignees

• Scripps Research Inst

Inventors

• Romesberg Floyd E
• Zhang Yorke

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C07K14/405 Primary

  from algae · 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

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N9/22

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