title: Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes.
abstract: Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.
authors.affiliation: Department of Chemistry, Franklin & Marshall College, Lancaster, Pennsylvania 17604-3003, USA.
authors.forename: Jason L
authors.initials: JL
authors.lastname: Seitchik
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Peeler
authors.forename: Michael T
authors.initials: MT
authors.lastname: Taylor
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Blackman
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Rhoads
authors.forename: Richard B
authors.initials: RB
authors.lastname: Cooley
authors.forename: Christian
authors.initials: C
authors.lastname: Refakis
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Fox
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002384:Catalysis; D002621:Chemistry / Q000379:methods*; D034242:Cyclooctanes / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017017:Methanococcus / Q000378:metabolism; D008956:Models, Chemical; D008968:Molecular Conformation; D011506:Proteins / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry
chemical_list: D000596:Amino Acids; D034242:Cyclooctanes; D011506:Proteins; D011725:Pyridines; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D014448:Tyrosine-tRNA Ligase