The mutual interaction network between all five PylRS classes based on the activity between the characterized PylRS enzymes and Pyl tRNAs. Mutually orthogonal pairs can be found using PylRS enzymes from classes A and B, classes A and S, classes B and S, classes C and N, and classes C and S (double-headed grey arrows). Therefore, five out of ten possible mutually orthogonal combinations were discovered; the other five combinations each showed one undesired cross-reactivity (single-headed red arrows). These orthogonal interactions were identified without engineering to tailor PylRS–tRNA Pyl interactions. For no two PylRS classes did all combinations of pairs possess two-sided cross-reactivity.
