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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