Non‐Canonical Amino Acids for Engineering Peptides and Proteins with new Functions

The universal genetic code, which specifies the 20 standard amino acids (AAs), forms the basis for all natural proteins. Researchers have developed efficient and robust in vivo and in vitro strategies to overcome the constraints of the genetic code to expand the repertoire of AA building blocks that can be ribosomally incorporated into proteins. This review summarizes the development of these in vivo and in vitro systems and their subsequent use for engineering of peptides and proteins with new functions. In vivo genetic code expansion employing engineered othogonal tRNA/aaRS pairs has led to the development of proteins that selectively bind small molecules, cleave nucleic acids and catalyze non‐natural chemical transformations. In vitro genetic code reprogramming using Flexizymes coupled with mRNA display has resulted in potent macrocyclic peptides that selectively bind to therapeutically important proteins. Through these examples, we hope to illustrate how genetic code expansion and reprogramming, especially when coupled with directed evolution or in vitro selection techniques, have emerged as powerful tools for expanding the functional capabilities of peptides and proteins.