The Design, Synthesis, and Evolution of Macrocyclic mRNA Display Libraries Containing Unnatural Amino Acids

Combinatorial design is a powerful method for generating ligands that disrupt protein-protein interactions, particularly in the absence of structural information. mRNA display is powerful in vitro biological display technique that enables the synthesis and evolution of trillion-member peptide libraries. Previously, mRNA display libraries were limited to linear peptides composed entirely from natural (protegenic) amino acids. This work describes the redesign of mRNA display libraries to include chemical functionalities that hitherto have remained the province of traditional chemical libraries and natural products. The incorporation of unnatural (nonproteogenic) amino acids through nonsense suppression and selection for the unnatural chemical functionality demonstrated that suppression technologies were compatible with biological display. The incorporation of N-methylated amino acids by sequential sense suppression was shown to generate unnatural oligomers with improved proteolytic resistance. Post-translational macrocyclization was shown to be an effective and general strategy to constrain displayed peptides without the use of redox-labile disulfide bonds. Cotranslational unnatural amino acid incorporation and post-translational macrocyclization were combined into a new technological platform and employed in the selection of a high-affinity cyclic ligand for Gαi1. This new technology represents a significant step toward the evolution of natural product-like compounds from trillion-member combinatorial libraries.