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We recently characterized a new class of pyrrolysyl-tRNA synthetase (PylRS)/ Pyl tRNA pairs from Methanomassiliicocales that are active and orthogonal in Escherichia coli. The aminoacyl-tRNA synthetases (aaRSs) of these pairs lack the N-terminal domain that is essential for tRNA recognition and in vivo activity in the Methanosarcina mazei ( Mm) PylRS but share a homologous active site with MmPylRS; this facilitates the transplantation of mutations discovered with existing PylRS systems into the new PylRS systems to reprogram their substrate specificity for the incorporation of noncanonical amino acids (ncAAs). Several of the new PylRS/ Pyl tRNA pairs, or their evolved variants [including Methanomethylophilus alvus ( Ma) PylRS/ Ma Pyl tRNA(6) CUA ], are mutually orthogonal to the MmPylRS/ Mm Pyl tRNA pair, and the active sites of the Mm pair and Ma pair can be diverged to enable the incorporation of distinct ncAAs in response to distinct codons via orthogonal translation in E. coli. Here we demonstrate that MaPylRS/ Ma Pyl tRNA(6) CUA is orthogonal to the aaRSs and tRNAs in mammalian cells and directs efficient incorporation of ncAAs into proteins. Moreover, we confirm that the MaPylRS/ Ma Pyl tRNA(6) and MmPylRS/ Mm Pyl tRNA pairs are mutually orthogonal in mammalian cells and demonstrates that these pairs can be used to encode distinct ncAAs into a protein in mammalian cells. Thus, the MaPylRS/ Ma Pyl tRNA(6) CUA pair provides an additional pair that is orthogonal in both E. coli and mammalian systems and is mutually orthogonal to the most widely used system for genetic code expansion. Our results provide a foundation for expanding the scope of genetic code expansion and may also facilitate strategies for proteome-wide ncAA tagging with mutually orthogonal systems.

An Evolved Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase/tRNA Pair Is Highly Active and Orthogonal in Mammalian Cells