Abstract
Genetic code expansion enables the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins both in vitro and in vivo. In addition to a widely applied nonsense suppression strategy, the use of quadruplet codons could further expand the genetic code. A general approach to genetically incorporate ncAAs in response to quadruplet codons is achieved by utilizing an engineered aminoacyl-tRNA synthetase (aaRS) together with a tRNA variant containing an expanded anticodon loop. Here we provide a protocol to decode quadruplet UAGA codon with a ncAA in mammalian cells. We also describe microscopy imaging and flow cytometry analysis of ncAA mutagenesis in response to quadruplet codons.
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Acknowledgments
This work was supported by the National Science Foundation (grant 1553041 to J.G.), National Institute of Health (grant 1R01GM138623 to J.G. and W.N.), and NIH National Institutes of General Medical Sciences (grant P20 GM113126 to J.G. and W.N.).
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Chen, Y., Gao, T., He, X., Niu, W., Guo, J. (2023). Genetic Code Expansion in Mammalian Cells Through Quadruplet Codon Decoding. In: Tsai, YH., Elsässer, S.J. (eds) Genetically Incorporated Non-Canonical Amino Acids. Methods in Molecular Biology, vol 2676. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3251-2_13
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DOI: https://doi.org/10.1007/978-1-0716-3251-2_13
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