Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons

Erome Daniel Hankore, Linyi Zhang, Yan Chen, Kun Liu, Wei Niu, Jiantao Guo

Research output: Contribution to journalArticlepeer-review

16 Scopus citations


Genetic incorporation of noncanonical amino acids has emerged as a powerful tool for the study of protein structure and function. While the three triplet nonsense codons have been widely explored, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acid mutagenesis. Here we demonstrated for the first time that two orthogonal quadruplet codons could be used to simultaneously encode two different noncanonical amino acids within a single protein in bacterial cells. To achieve this, we fine-tuned the interaction between aminoacyl-tRNA synthetase and tRNA, which afforded up to 21-fold improvement in quadruplet codon decoding efficiency. This work represents a significant step toward the use of multiple quadruplet codons for noncanonical amino acid mutagenesis. Simultaneous incorporation of two or more noncanonical amino acids is of significant importance for biological applications that can benefit from multiple unique functional groups, such as fluorescence resonance energy transfer and nuclear magnetic resonance studies, and ultimately for the synthesis of completely unnatural biopolymers as new biomaterials.

Original languageEnglish (US)
Pages (from-to)1168-1174
Number of pages7
JournalACS Synthetic Biology
Issue number5
StatePublished - May 17 2019


  • four-base codon
  • genetic code
  • orthogonal codon
  • quadruplet codon
  • unnatural amino acids

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)


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