biostudies-literatureChen YNIHNIGMS NIH HHSNational Science Foundatione94https://www.ebi.ac.uk/biostudies/studies/S-EPMC9458425biostudies-literatureUnknown50(16)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 ofNucleic acids researchNoncanonical amino acid mutagenesis in response to recoding signal-enhanced quadruplet codons.PMC94584251R01GM138623MCB 1553041P20 GM113126R01 GM138623Ma BLiu KGuo JHe XChen YNiu WGao TfalseNoncanonical amino acid mutagenesis in response to recoding signal-enhanced quadruplet codons.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 of2022-01-01T00:00:00Z2022 Sep2025-04-05T10:25:29.873Z2025-04-05T10:25:29.873ZS-EPMC94584253565709410.1093/nar/gkac474