Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs

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/(Pyl)tRNA pair from Methanosarcina 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(Pyl)RNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/Mm(Pyl)RNA pair has proved challenging. Here we demonstrate that several Delta NPyIRS/(Pyl)RNA(CUA) pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/(Pyl)RNA pairs that are mutually orthogonal to the MmPyIRS/Mm(Pyl)RNA 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/(Pyl)RNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.