A Selection of Macrocyclic Peptides That Bind STING From an mRNA‐Display Library With Split Degenerate Codons

Recent improvements in mRNA display have enabled the selection of peptides that incorporate non‐natural amino acids, thus expanding the 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 that 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. In vitro selection of macrocyclic peptides from a library that uses a 25‐amino‐acid alphabet has allowed the identification of peptides that block binding of human STING to its natural ligand and induce dimerization of STING monomers. Our method splits multiple degenerate codons and reassigns them to non‐natural amino acids.