Novel nucleoside triphosphate transporter and uses thereof

Patent metadata

Field	Value
Publication number	US-2020277342-A1
Application number	US-202016839741-A
Country	US
Kind code	A1
Filing date	Apr 3, 2020
Priority date	Jun 24, 2016
Publication date	Sep 3, 2020
Grant date	—

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 a cell, comprising:
(a) expressing in the cell a recombinant nucleoside triphosphate transporter, wherein the recombinant nucleoside triphosphate transporter comprises an amino acid sequence comprising a deletion, (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.

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 recombinant nucleoside triphosphate transporter is from Phaeodactylum tricornutum.

5 . The method of claim 1 , wherein the deletion comprises about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 65, 66, 70, or more amino acid residues.

6 . The method of claim 1 , wherein the deletion is a N-terminal deletion.

7 . The method of claim 1 , wherein the amino acid sequence of the recombinant nucleoside triphosphate transporter is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% identical to SEQ ID NO: 4.

8 . The method of claim 1 , wherein the cell is an engineered cell.

9 . The method of claim 8 , wherein the engineered cell is E. coli.

10 . The method of claim 1 , wherein the cell is a prokaryotic cell.

11 . The method of claim 10 , wherein the cell is E. coli.

12 . The method of claim 1 , wherein the nucleic acid molecule comprises one or more unnatural nucleotides comprising a base selected from the group consisting of

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

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

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

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

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

18 . A method of increasing production of a DNA nucleic acid molecule in a cell comprising:
(a) expressing in the cell a recombinant nucleoside triphosphate transporter, wherein the recombinant nucleoside triphosphate transporter comprises an amino acid sequence comprising a deletion, (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:

19 . The method of claim 18 , wherein the cell is E. coli.

20 . The method of claim 18 , wherein the recombinant nucleoside triphosphate transporter is from Phaeodactylum tricornutum.

21 . The method of claim 18 , wherein the deletion comprises about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 65, 66, 70, or more amino acid residues.

22 . The method of claim 18 , wherein the deletion is a N-terminal deletion.

23 . The method of claim 18 , wherein the amino acid sequence of the recombinant nucleoside triphosphate transporter is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% identical to SEQ ID NO: 4.

24 . A method of increasing production of a RNA nucleic acid molecule in a cell, comprising:
(a) expressing in the cell a recombinant nucleoside triphosphate transporter, wherein the recombinant nucleoside triphosphate transporter comprises an amino acid sequence comprising a deletion, (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:

25 . The method of claim 24 , wherein the cell is E. coli.

26 . The method of claim 24 , wherein the recombinant nucleoside triphosphate transporter is from Phaeodactylum tricornutum.

27 . The method of claim 24 , wherein the deletion comprises about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 65, 66, 70, or more amino acid residues.

28 . The method of claim 24 , wherein the deletion is a N-terminal deletion.

29 . The method of claim 24 , wherein the amino acid sequence of the recombinant nucleoside triphosphate transporter is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% identical to SEQ ID NO: 4.

Assignees

• Scripps Research Inst

Inventors

• Romesberg Floyd E
• Zhang Yorke

Classifications

• C12N9/22

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

• C07K14/405 Primary

  from algae · CPC title

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · 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