Using macrocyclic peptides as specific drug-like inhibitors has many advantages over using linear equivalents, including conformational rigidity in the unbound state, protease resistance and cell permeability. These drug-like properties can be further improved by incorporating unnatural amino acids into the peptide chain, for example, D stereochemistry, unusual side chains and N-methylation. Using an in vitro evolved ribozyme that is capable of charging any amino acid onto any tRNA, a reconstituted 'PURE' in vitro translation system, and the ability to screen trillions of peptide candidates using mRNA display, the Suga lab has developed a fast route to discovering macrocyclic peptide binders for particular protein targets. Here this system is applied to generate macrocyclic, N-methyl containing peptides that bind to a human ubiquitin protease.