Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNA(Pyl) pairs

Mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs provide a foundation for encoding non-canonical amino acids (ncAAs) into proteins, and encoded non-canonical polymer and macrocycle synthesis. Here we discover quintuply orthogonal pyrrolysyl-tRNA synthetase (PylRS)/ 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 (existing classes: +N, A, B, and newly defined classes: C, S). Most PylRS clusters belong to classes that are unexplored for orthogonal pair generation. By testing pairs from distinct clusters and classes, and pyl 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, 1324 triply orthogonal pairs, 128 quadruply orthogonal pairs, and 8 quintuply orthogonal pairs. These advances may provide a key foundation for encoded polymer synthesis.