Non-canonical amino acids have been introduced to the lasso peptide microcin J25.

Pyrrolysine is the 22nd proteinogenic amino acid encoded into proteins in response to amber (TAG) codons in a small number of archaea and bacteria. The incorporation of pyrrolysine is facilitated by a specialized aminoacyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNA^Pyl). The secondary structure of tRNA^Pyl contains several unique features not found in canonical tRNAs. Numerous studies have demonstrated that the PylRS/tRNA^Pyl pair from archaea is orthogonal in E. coli and eukaryotic hosts, which has led to the widespread use of this pair for the genetic incorporation of non-canonical amino acids. In this brief review we examine the work that has been done to elucidate the structure of tRNA^Pyl, its interaction with PylRS, and survey recent progress on the use of tRNA^Pyl as a tool for genetic code expansion.

Seven phenylalanine derivatives with small ortho substitutions were genetically encoded in Escherichia coli and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNA_CUA^Pyl. Ortho substitutions of the phenylalanine derivatives reported herein include three halides, methyl, methoxy, nitro, and nitrile. These compounds have the potential for use in multiple biochemical and biophysical applications. Specifically, we demonstrated that o-cyano-phenylalanine could be used as a selective sensor to probe the local environment of proteins and applied this to study protein folding/unfolding. For six of these compounds this constitutes the first report of their genetic incorporation in living cells. With these compounds the total number of substrates available for PylRS(N346A/C348A) is increased to nearly 40, which demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine with a substitution at any side-chain aromatic position as a substrate. To our knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase with such a high substrate promiscuity.