Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids.

Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNAPyl pairs and cross recognition between nonsense codons and various tRNAPyl anticodons in the Escherichia coli BL21(DE3) cell strain are reported. is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, Nε-tert-butoxycarbonyl-l-lysine (BocK). Similar to, is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS- pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS- pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.

Detailed kinetic analyses of inverse electron-demand Diels–Alder cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition reactions were conducted and the reactions were applied for rapid protein bioconjugation. When reacted with a tetrazine or a diaryl nitrilimine, strained alkene/alkyne entities including norbornene, trans-cyclooctene, and cyclooctyne displayed rapid kinetics. To apply these “click” reactions for site-specific protein labeling, five tyrosine derivatives that contain a norbornene, trans-cyclooctene, or cyclooctyne entity were genetically encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNACUAPyl pair. Proteins bearing these noncanonical amino acids were successively labeled with a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro and in living cells.