RESUMEN
Labeling of biomolecules in live eukaryotic cells has been limited by low component stability and slow reaction rates. We show that genetically encoded tetrazine amino acids in proteins reach reaction rates of 8 × 104 M-1 s-1 with sTCO reagents, making them the fastest site-specific bioorthogonal labels in eukaryotic systems. We demonstrate that tetrazine amino acids are stable on proteins and are capable of quantitative labeling with sTCO reagents. The exceptionally high reaction rate of this ligation minimizes label concentration, allowing for substoichiometric in vivo eukaryotic protein labeling where the concentration of the label is less than the concentration of the protein. This approach offers unprecedented control over the composition and stability of the protein tag. We anticipate that this system will have a broad impact on labeling and imaging studies because it can be used where all generally orthogonal PylRS/tRNA pairs are employed.