Reagents and methods for bioorthogonal labeling of biomolecules in living cells

A method for making a protein or a polypeptide, comprising:

• culturing a cell in a medium including a tetrazine amino acid, the tetrazine amino acid comprising an amino acid bound directly to a tetrazine via a carbon link, wherein the cell includes:
• a nucleic acid that encodes a protein or a polypeptide and comprises a selector codon;
• an orthogonal tRNA that is specific for the tetrazine amino acid and that recognizes an orthogonal Methanococcus jannaschii (Mj) aminoacyl-tRNA synthetase that is specific for the tetrazine amino acid and recognizes the selector codon, and
• the orthogonal Mj aminoacyl-tRNA synthetase aminoacylates the orthogonal tRNA with the tetrazine amino acid;
• and
• in the cultured cell and using the orthogonal Mj aminoacyl-tRNA synthetase and the orthogonal tRNA, incorporating the tetrazine amino acid into the protein or the polypeptide to provide a protein or polypeptide having a tetrazine amino acid residue bound to the protein or the polypeptide, wherein the tetrazine amino acid is selected from the group consisting of 4-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0-methyl), 4-(6-ethyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0-ethyl), 4-(6-isopropyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0-isopropyl), and 4-(6-butyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0-n-butyl), wherein the orthogonal Mj aminoacyl-tRNA synthetase comprises modified amino acids at positions 32, 65, 108, 109, 158, and 162, and wherein the modified amino acids are:
• Ala or Gly at position 32;
• Ala, Ser, Val, or Gln at position 65;
• Glu, Gln, His, Asp, Ser, Leu, or Ala at position 108;
• His, Glu, Ser, Asp, or Pro at position 109;
• Gly, Ser, or Asn at position 158;
• and
• Ser, Ala, Gly, or Asn at position 162.