Abstract
The ability to introduce non-canonical amino acids into peptides and proteins is facilitated by working within in vitro translation systems. Non-canonical amino acids can be introduced into these systems using sense codon reprogramming, stop codon suppression, and by breaking codon degeneracy. Here, we review how these techniques have been used to create proteins with novel properties and how they facilitate sophisticated studies of protein function. We also discuss how researchers are using in vitro translation experiments with non-canonical amino acids to explore the tolerance of the translation apparatus to artificial building blocks. Finally, we give several examples of how non-canonical amino acids can be combined with mRNA-displayed peptide libraries for the creation of protease-stable, macrocyclic peptide libraries for ligand discovery.