Incorporation of unnatural nucleotides and methods thereof

Patent metadata

Field	Value
Publication number	US-11834689-B2
Application number	US-202117196151-A
Country	US
Kind code	B2
Filing date	Mar 9, 2021
Priority date	Jul 11, 2017
Publication date	Dec 5, 2023
Grant date	Dec 5, 2023

Abstract

Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.

First claim

What is claimed is:

1. A cell comprising:
a. a nucleoside triphosphate transporter from Phaeodactylum tricornutum;
b. a tRNA from Methanosarcina mazei or Methanococcus jannaschii comprising an anticodon, wherein the anticodon comprises a sequence selected from GGY, GYG, YGG, GAY, GYA, YGA, GCY, GYC, YGC, GUY, GYU, YGU, CAY, CYA, YCA, CGY, CYG, YCG, CUY, CYU, YCU, CCY, CYC, YCC, AAY, AYA, YAA, AGY, AYG, YAG, ACY, AYC, YAC, AUY, AYU, YAU, UUY, UYU, YUU, UAY, UYA, YUA, UGY, UYG, YUG, UCY, UYC, YUC, GYY, YGY, YYG, CYY, YCY, YYC, AYY, YAY, YYA, UYY, YUY, YYU, YYY, GGX, GXG, XGG, GAX, GXA, XGA, GCX, GXC, XGC, GUX, GXU, XGU, CAX, CXA, XCA, CGX, CXG, XCG, CUX, CXU, XCU, CCX, CXC, XCC, AAX, AXA, XAA, AGX, AXG, XAG, ACX, AXC, XAC, AUX, AXU, XAU, UUX, UXU, XUU, UAX, UXA, XUA, UGX, UXG, XUG, UCX, UXC, XUC, GXX, XGX, XXG, CXX, XCX, XXC, AXX, XAX, XXA, UXX, XUX, XXU, or XXX, and wherein each of Y and X are an unnatural nucleotide comprising an unnatural nucleobase selected from

c. an aminoacyl tRNA synthetase from Methanosarcina or Methanococcus jannaschii ; and
d. an mRNA comprising a codon, wherein the codon comprises a sequence selected from GGY, GYG, YGG, GAY, GYA, YGA, GCY, GYC, YGC, GUY, GYU, YGU, CAY, CYA, YCA, CGY, CYG, YCG, CUY, CYU, YCU, CCY, CYC, YCC, AAY, AYA, YAA, AGY, AYG, YAG, ACY, AYC, YAC, AUY, AYU, YAU, UUY, UYU, YUU, UAY, UYA, YUA, UGY, UYG, YUG, UCY, UYC, YUC, GYY, YGY, YYG, CYY, YCY, YYC, AYY, YAY, YYA, UYY, YUY, YYU, YYY, GGX, GXG, XGG, GAX, GXA, XGA, GCX, GXC, XGC, GUX, GXU, XGU, CAX, CXA, XCA, CGX, CXG, XCG, CUX, CXU, XCU, CCX, CXC, XCC, AAX, AXA, XAA, AGX, AXG, XAG, ACX, AXC, XAC, AUX, AXU, XAU, UUX, UXU, XUU, UAX, UXA, XUA, UGX, UXG, XUG, UCX, UXC, XUC, GXX, XGX, XXG, CXX, XCX, XXC, AXX, XAX, XXA, UXX, XUX, XXU, or XXX, and wherein each of Y and X are an unnatural nucleotide comprising an unnatural nucleobase selected from

and wherein the anticodon of the tRNA pairs with the codon of the mRNA.

2. The cell of claim 1 , wherein the cell further comprises an oligonucleotide encoding the tRNA.

3. The cell of claim 1 , wherein the cell further comprises an oligonucleotide encoding the aminoacyl tRNA synthetase.

4. The cell of claim 1 , wherein the cell further comprises an oligonucleotide encoding the mRNA.

5. The cell of claim 1 , wherein the cell further comprises an oligonucleotide that encodes the tRNA and the mRNA.

6. The cell of claim 1 , wherein the cell further comprises an oligonucleotide that encodes the tRNA, the mRNA, and the aminoacyl tRNA synthetase.

7. The cell of claim 1 , wherein the unnatural nucleobase in the anticodon is

and the unnatural nucleobase in the codon is

8. The cell of claim 7 , wherein the unnatural nucleobase in the codon is

and the unnatural nucleobase in the anticodon is

9. The cell of claim 1 , wherein the anticodon comprises the sequence GYT or GYC, wherein Y comprises the unnatural nucleobase.

10. The cell of claim 9 , wherein the unnatural nucleobase of Y is

11. The cell of claim 10 , wherein the unnatural nucleobase of Y is

12. The cell of claim 10 , wherein the unnatural nucleobase of Y is

13. The cell of claim 1 , wherein the codon comprises the sequence AXC or GXC, wherein X comprises the unnatural nucleobase.

14. The cell of claim 13 , wherein the codon comprises the sequence AXC.

15. The cell of claim 14 , wherein the unnatural nucleobase of X is

16. The cell of claim 14 , wherein the unnatural nucleobase of X is

17. The cell of claim 13 , wherein the codon comprises the sequence GXC.

18. The cell of claim 17 , wherein the unnatural nucleobase of X is

19. The cell of claim 17 , wherein the unnatural nucleobase of X is

20. The cell of claim 1 , wherein the aminoacyl tRNA synthetase is from Methanosarcina barkeri.

21. The cell of claim 1 , wherein the aminoacyl tRNA synthetase is from Methanococcus jannaschii.

22. The cell of claim 1 , wherein the aminoacyl tRNA synthetase is a pyrrolysyl tRNA synthetase.

23. The cell of claim 1 , wherein the aminoacyl tRNA synthetase is a tyrosyl tRNA synthetase.

24. The cell of claim 1 , wherein the cell is a microorganism or bacterium.

25. The cell of claim 1 , wherein the cell is an E. coli cell.

Assignees

• Synthorx Inc
• Scripps Research Inst

Inventors

• Ptacin Jerod
• Caffaro Carolina
• Aerni Hans
• Zhang Yorke
• Fischer Emil C
• Feldman Aaron W
• Dien Vivian T
• Romesberg Floyd E

Classifications

• C12N9/93 Primary

  Ligases (6) · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12Y601/01026

  Pyrrolysine-tRNAPyl ligase (6.1.1.26) · CPC title

• C12N15/11 Primary

  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