US 12,319,944 B2
Incorporation of unnatural nucleotides and methods thereof
Jerod Ptacin, La Jolla, CA (US); Carolina Caffaro, La Jolla, CA (US); Hans Aerni, La Jolla, CA (US); Yorke Zhang, La Jolla, CA (US); Emil C. Fischer, La Jolla, CA (US); Aaron W. Feldman, La Jolla, CA (US); Vivian T. Dien, La Jolla, CA (US); and Floyd E. Romesberg, La Jolla, CA (US)
Assigned to THE SCRIPPS RESEARCH INSTITUTE, La Jolla, CA (US); and SYNTHORX, INC., Bridgewater (NL)
Filed by Synthorx, Inc., Cambridge, MA (US); and The Scripps Research Institute, La Jolla, CA (US)
Filed on Oct. 4, 2023, as Appl. No. 18/481,086.
Application 18/481,086 is a division of application No. 17/196,151, filed on Mar. 9, 2021, granted, now 11,834,689.
Application 17/196,151 is a continuation of application No. 16/629,255, abandoned, previously published as PCT/US2018/041509, filed on Jul. 10, 2018.
Claims priority of provisional application 62/531,325, filed on Jul. 11, 2017.
Prior Publication US 2024/0043823 A1, Feb. 8, 2024
Int. Cl. C12N 9/00 (2006.01); C12N 15/113 (2010.01); C12N 15/90 (2006.01); C12P 21/02 (2006.01)
CPC C12N 9/93 (2013.01) [C12N 15/113 (2013.01); C12N 15/90 (2013.01); C12P 21/02 (2013.01); C12Y 601/01026 (2013.01)] 28 Claims
 
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:

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(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:

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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.