Selection of macrocyclic peptides that bind STING from an mRNA-display library with split degenerate codons.

Recent improvements in mRNA display have enabled selection of peptides that incorporate non-natural amino acids, thus expanding chemical diversity of macrocycles beyond what is accessible in nature. Such libraries have incorporated non-natural amino acids at the expense of natural amino acids, by reassigning their codons. Here we report an alternative approach to expanded amino-acid diversity, which preserves all 19 natural amino acids (no methionine) and adds 6 non-natural amino acids, resulting in the highest sequence complexity reported to date. We have applied mRNA display to this 25-letter library to select functional macrocycles that bind human STING, a protein involved in immunoregulation. The resulting STING-binding peptides include a 9-mer macrocycle with a dissociation constant (KD) of 3.4 nM, which blocks binding of cGAMP to STING and induces STING dimerization. This approach is generalizable to expanding the amino-acid alphabet in a library beyond 25 building blocks.