Advances in the mechanism and understanding of site-selective noncanonical amino acid incorporation

There are many approaches to introduce non-native functionality into proteins either translationally or post-translationally. When a noncanonical amino acid (NAA) is incorporated translationally, the host organism's existing translational machinery is relied upon to insert the amino acid by the same well-established mechanisms used by the host to achieve high fidelity insertion of its canonical amino acids. Research into the in vivo incorporation of NAAs has typically concentrated on evolving or engineering aminoacyl tRNA synthetases (aaRSs); however, new studies have increasingly focused on other members of the translational apparatus, for example entire ribosomes, in attempts to increase the fidelity and efficiency of incorporation of ever more structurally diverse NAAs. As the biochemical methods of NAA systems increase in complexity, it is informative to ask whether the ‘rules’ for canonical translation (i.e. aaRSs, tRNA, ribosomes, elongation factors, amino acid uptake, and metabolism) hold for NAA systems, or whether new rules are warranted. Here, recent advances in introducing novel chemical functionality into proteins are highlighted.

► Important topics in NAA biological chemistry are: chemical functionality, fidelity, and efficiency.
► Tyrosyl and pyrrolysyl enzymes are amendable to an increasing array of noncanonical amino acids (NAAs).
► Advances in the biology of NAAs show common and uncommon themes with their canonical counterparts.
► Recent studies show the extent to which the translation can be engineered for NAA incorporation.