Incorporation of backbone modifications in mRNA-displayable peptides

Here we comprehensively summarize the most recent efforts in our research team, aiming at installing N-methyl and azole backbones into peptides expressed in translation. The genetic code reprogramming using the Flexible In-vitro Translation system (FIT system) has proven to be the most reliable and versatile approach for ribosomally installing various exotic amino acids. However, it had been yet difficult in translating diverse kinds of multiple and consecutive sequences of N-methyl amino acids (^MeAAs). We have recently reported that a semi-rational fine tuning of ^MeAA-tRNA affinities for EF-Tu by altering tRNA T-stem sequence achieves efficient delivery of ^MeAA-tRNAs to the ribosome. Indeed, this approach has made it possible to express N-methyl-peptides containing multiple ^MeAAs with a remarkably high fidelity. Another interesting backbone modification in peptides is azole moieties often found in natural products, but they are explicitly installed by post-translational modifying enzymes. We have recently devised a method to bypass such enzymatic processes where a bromovinyl group-containing amino acid is incorporated into the peptide by genetic code reprogramming and then chemically converted to an azole group via an intramolecular heterocyclization reaction. These methods will grant more drug-like properties to peptides than ordinary peptides in terms of protease resistance and cell membrane permeability. Particularly when they can be integrated with in vitro mRNA display, such as the RaPID system, the discovery of de novo bioactive peptides can be realized.