Incorporation of unnatural nucleotides and methods thereof

Patent metadata

Field	Value
Publication number	US-12319944-B2
Application number	US-202318481086-A
Country	US
Kind code	B2
Filing date	Oct 4, 2023
Priority date	Jul 11, 2017
Publication date	Jun 3, 2025
Grant date	Jun 3, 2025

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 method of producing a protein comprising an unnatural amino acid in a cell, the method comprising:
contacting the cell with an unnatural amino acid, the cell comprising:
(i) a nucleoside triphosphate transporter from Phaeodactylum tricornutum;
(ii) a tRNA from Methanosarcina mazei or Methanococcus jannaschii comprising an anticodon, the anticodon comprising an unnatural nucleotide comprising a first unnatural nucleobase independently selected from:

(iii) an aminoacyl tRNA synthetase characterized in that it aminoacylates the tRNA with the unnatural amino acid;
(iv) an mRNA encoding the protein comprising the unnatural amino acid and comprising a codon, the codon comprising an unnatural nucleotide comprising a second unnatural nucleobase independently selected from:

wherein the anticodon of the tRNA pairs with the codon of the mRNA;
wherein the mRNA is translated in the cell involving the tRNA, thereby producing a protein comprising the unnatural amino acid.

2. The method of claim 1 , further comprising introducing an unnatural nucleotide into the cell via the nucleoside triphosphate transporter.

3. The method of claim 1 , wherein the first unnatural nucleobase or the second unnatural nucleobase is

4. The method of claim 1 , wherein the second unnatural nucleobase is

and the first unnatural nucleobase is

5. The method of claim 1 , wherein the anticodon comprises the sequence GYU or GYC, wherein Y is an unnatural nucleotide comprising the first unnatural nucleobase.

6. The method of claim 5 , wherein Y is an unnatural nucleotide comprising the first unnatural nucleobase, which is

7. The method of claim 1 , wherein the aminoacyl tRNA synthetase is from Methanosarcina barkeri or Methanococcus jannaschii.

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

9. The method of claim 1 , wherein the cell is E. coli.

10. A method of producing a protein comprising an unnatural amino acid in a cell, the method comprising contacting the cell with at least one unnatural nucleotide and at least one unnatural amino acid, the cell comprising:
(i) a nucleoside triphosphate transporter from Phaeodactylum tricornutum;
(ii) a tRNA from Methanosarcina mazei or Methanococcus jannaschii comprising an anticodon, the anticodon comprising an unnatural nucleotide comprising a first unnatural nucleobase independently selected from:

(iii) an aminoacyl tRNA synthetase characterized in that it aminoacylates the tRNA with the unnatural amino acid;
(iv) a nucleic acid encoding an mRNA encoding the protein comprising the unnatural amino acid and comprising a codon, the codon comprising an unnatural nucleotide comprising a second unnatural nucleobase independently selected from:

wherein the anticodon of the tRNA pairs with the codon of the mRNA
wherein:
(a) the at least one unnatural nucleotide independently comprises the first nucleobase or the second nucleobase:

(b)(i) the mRNA comprising the codon is synthesized by transcription and the at least one unnatural nucleotide comprising the second nucleobase is incorporated into the mRNA during transcription; and/or (ii) the tRNA comprising the anticodon is synthesized by transcription and the at least one unnatural nucleotide comprising the first nucleobase is incorporated into the tRNA during transcription; and
(c) the mRNA is translated into the cell involving the tRNA, thereby producing a protein comprising the unnatural amino acid.

11. The method of claim 10 , wherein the at least one unnatural nucleotide is introduced into the cell via the nucleoside triphosphate transporter and is incorporated into the mRNA and/or the tRNA during transcription.

12. The method of claim 10 , wherein the first unnatural nucleobase or the second unnatural nucleobase of the unnatural nucleotide in the anticodon or the codon is

and/or wherein the unnatural nucleobase of the unnatural nucleotide in the mRNA is

and the unnatural nucleobase of the unnatural nucleotide in the tRNA is

and/or wherein the anticodon comprises the sequence GYU or GYC, wherein Y is an unnatural nucleotide comprising the first unnatural nucleobase.

13. The method of claim 12 , wherein Y is an unnatural nucleotide comprising the first unnatural nucleobase, which is

14. The method of claim 10 , wherein the cell is a microorganism or bacterium.

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

16. The method of claim 1 , wherein at least one or both of the first unnatural nucleobase and the second unnatural nucleobase are each independently selected from:

17. The method of claim 1 , wherein at least one or both of the first unnatural nucleobase and the second unnatural nucleobase are each independently selected from:

18. The method of claim 1 , wherein the codon comprises the sequence AXC or GXC, wherein X is an unnatural nucleotide comprising the second unnatural nucleobase.

19. The method of claim 18 , wherein X is an unnatural nucleotide comprising the second unnatural nucleobase, which is

20. The method of claim 1 , wherein the unnatural amino acid is p-azido-phenylalanine (pAzF) or N 6 -[(2-propynyloxy) carbonyl]-l-lysine (PrK).

21. The method of claim 10 , further comprising expressing the aminoacyl tRNA synthetase from a plasmid in the ce

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

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12Y601/01026

  Pyrrolysine-tRNAPyl ligase (6.1.1.26) · CPC title

• C12P21/02 Primary

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

• C12N9/93 Primary

  Ligases (6) · CPC title