While amber suppression is the most common approach to introduce noncanonical amino acids into proteins in live cells, quadruplet codon decoding has potential to enable a greatly expanded genetic code with up to 256 new codons for protein biosynthesis. Since triplet codons are the predominant form of genetic code in nature, quadruplet codon decoding often displays limited efficiency. In this work, we exploited a new approach to significantly improve quadruplet UAGN and AGGN (N = A, U, G, C) codon decoding efficiency by using recoding signals imbedded in mRNA. With representative recoding signals, the expression level of mutant proteins containing UAGN and AGGN codons reached 48% and 98% of that of the wild-type protein, respectively. Furthermore, this strategy mitigates a common concern of reading-through endogenous stop codons with amber suppression-based system. Since synthetic recoding signals are rarely found near the endogenous UAGN and AGGN sequences, a low level of undesirable suppression is expected. Our strategy will greatly enhance the utility of noncanonical amino acid mutagenesis in live-cell studies.
more » « less- NSF-PAR ID:
- 10370946
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 16
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. e94-e94
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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