Beyond the canonical 20 amino acids: expanding the genetic lexicon

Abstract

The ability to genetically encode unnatural amino acids beyond the common 20 has allowed unprecedented control over the chemical structures of recombinantly expressed proteins. Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have been used together with nonsense, rare, or 4-bp codons to incorporate >50 unnatural amino acids into proteins in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and mammalian cell lines. This has allowed the expression of proteins containing amino acids with novel side chains, including fluorophores, post-translational modifications, metal ion chelators, photocaged and photocross-linking moieties, uniquely reactive functional groups, and NMR, IR, and x-ray crystallographic probes.

FIGURE 1.

Orthogonal aaRS/tRNA_CUA pair and translational machinery. The orthogonal aaRS (red) aminoacylates the orthogonal tRNA_CUA (blue, red CUA) with an unnatural amino acid (blue X) and does not cross-react with the endogenous aaRS/tRNA pairs (shown is the TyrRS (gray)/tRNA_AUA (black) pair). Aminoacyl-tRNA_CUA travels to the ribosome (brown), where it incorporates the unnatural amino acid in response to the UAG codon (red UAG).

FIGURE 2.

Unnatural amino acids and their applications. a, orthogonal chemistries enable the site-specific modification of proteins with a wide variety of molecules. b, examples of unnatural amino acids described in text.