Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

We recently described an orthogonal initiator tRNA (itRNATy2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here, we report a mutant of itRNATy2 (itRNATy2AUA) efficiently translation UAU tyrosine codon, giving rise proteins ncAA at their N-terminus. show that, cells expressing itRNATy2AUA, function as dual-use codon selectively encodes ncAAs initiating position and predominantly elongating positions. Using conjunction its cognate tyrosyl-tRNA synthetase two mutually pyrrolysyl-tRNA synthetases, demonstrate be reassigned along or UAA encode distinct same protein. Furthermore, by engineering substrate specificity one developed triply system enables simultaneous reassignment UAU, UAG, produce containing three precisely defined sites. To showcase utility this system, produced ncAAs, unique bioorthogonal functional groups, these separately modified multiple fluorescent probes.