title: Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid.
abstract: Bioorthogonal reactions for labeling biomolecules in live cells have been limited by slow reaction rates or low component selectivity and stability. Ideal bioorthogonal reactions with high reaction rates, high selectivity, and high stability would allow for stoichiometric labeling of biomolecules in minutes and eliminate the need to wash out excess labeling reagent. Currently, no general method exists for controlled stoichiometric or substoichiometric labeling of proteins in live cells. To overcome this limitation, we developed a significantly improved tetrazine-containing amino acid (Tet-v2.0) and genetically encoded Tet-v2.0 with an evolved aminoacyl-tRNA synthetase/tRNA(CUA) pair. We demonstrated in cellulo that protein containing Tet-v2.0 reacts selectively with cyclopropane-fused trans-cyclooctene (sTCO) with a bimolecular rate constant of 72,500 ± 1660 M(-1) s(-1) without reacting with other cellular components. This bioorthogonal ligation of Tet-v2.0-protein reacts in cellulo with substoichiometric amounts of sTCO-label fast enough to remove the labeling reagent from media in minutes, thereby eliminating the need to wash out label. This ideal bioorthogonal reaction will enable the monitoring of a larger window of cellular processes in real time.
authors.affiliation: §Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Blizzard
authors.affiliation: §Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
authors.forename: Dakota R
authors.initials: DR
authors.lastname: Backus
authors.affiliation: §Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
authors.forename: Wes
authors.initials: W
authors.lastname: Brown
authors.affiliation: §Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Bazewicz
authors.affiliation: ‡Brown Laboratory, Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: §Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
mesh_terms: D000596:Amino Acids / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics; D001671:Biochemistry / Q000379:methods*; D003001:Cloning, Molecular; D034242:Cyclooctanes / Q000737:chemistry; D003521:Cyclopropanes / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D063747:Methanocaldococcus / Q000201:enzymology; D010649:Phenylalanine / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D000596:Amino Acids; D034242:Cyclooctanes; D003521:Cyclopropanes; D006573:Heterocyclic Compounds, 1-Ring; D011506:Proteins; D012235:Rhodamines; D049452:Green Fluorescent Proteins; D010649:Phenylalanine; C030797:cyclopropane; D000604:Amino Acyl-tRNA Synthetases