Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs

Abstract

Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/ ^PyltRNA pair from M. mazei ( Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/ Mm ^PyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/ Mm ^PyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/ ^PyltRNA _CUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in E. coli. We create new PylRS/ ^PyltRNA pairs that are mutually orthogonal to the MmPylRS/ Mm ^PyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally we show that distinct PylRS/ ^PyltRNA derived pairs can function in the same cell, decode distinct codons, and incorporate distinct ncAAs.