Abstract
Mutually orthogonal aminoacyl transfer RNA synthetase/transfer RNA pairs provide a foundation for encoding non-canonical amino acids into proteins, and encoded non-canonical polymer and macrocycle synthesis. Here we discover quintuply orthogonal pyrrolysyl-tRNA synthetase (PylRS)/pyrrolysyl-tRNA (tRNA(Pyl)) pairs. We discover empirical sequence identity thresholds for mutual orthogonality and use these for agglomerative clustering of PylRS and tRNA(Pyl) sequences; this defines numerous sequence clusters, spanning five classes of PylRS/tRNA(Pyl) pairs (the existing classes +N, A and B, and newly defined classes C and S). Most of the PylRS clusters belong to classes that were unexplored for orthogonal pair generation. By testing pairs from distinct clusters and classes, and pyrrolysyl-tRNAs with unusual structures, we resolve 80% of the pairwise specificities required to make quintuply orthogonal PylRS/tRNA(Pyl) pairs; we control the remaining specificities by engineering and directed evolution. Overall, we create 924 mutually orthogonal PylRS/tRNA(Pyl) pairs, 1,324 triply orthogonal pairs, 128 quadruply orthogonal pairs and 8 quintuply orthogonal pairs. These advances may provide a key foundation for encoded polymer synthesis.