Directed Evolution of the Pyrrolysyl tRNA/aminoacyl tRNA Synthetase Pair for Rapid Evaluation of Sense Codon Reassignment Potential.

Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to codons in Predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the pyrrolysyl orthogonal tRNA/aaRS pair that activates and incorporates tyrosine. The evolved tRNA/aaRS pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating orthogonal pair. The new orthogonal pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved machinery are compared with related measurements employing the orthogonal pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the tyrosyl and pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different orthogonal pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the system will highlight the most promising positions at which the orthogonal pair may infiltrate the genetic code.