Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, *

Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyltransfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/ Pyl tRNA pairs are the most widely used system for genetic code expansion. Here we reveal that the sequences of ΔNPylRS/ ΔNPyl tRNA pairs (which lack Nterminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ΔNPyl tRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ΔNPyl tRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ ΔNPyl tRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/ Pyl tRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.

Bioactive naturally occurring macrocyclic peptides often exhibit a strong bias for hydrophobic residues. Recent advances in in vitro display technologies have made possible the identification of potent macrocyclic peptide ligands to protein targets of interest. However, such approaches have so far been restricted to using libraries composed of peptides containing mixtures of hydrophobic and hydrophilic/charged amino acids encoded by the standard genetic code. In the present study, we have demonstrated ribosomal expression of exotic macrocyclic peptides under a radically reprogrammed, relatively hydrophobic, genetic code, comprising 12 proteinogenic and 11 nonproteinogenic amino acids. Screening of this library for affinity to the interleukin-6 receptor (IL6R) as a case study successfully identified exotic macrocyclic peptide ligands with high affinity, validating the feasibility of this approach for the discovery of relatively hydrophobic exotic macrocyclic peptide ligands.