title: Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction.
abstract: The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNA(CUA) pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit 'turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Lang
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
authors.forename: Lloyd
authors.initials: L
authors.lastname: Davis
authors.affiliation: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Torres-Kolbus
authors.affiliation: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA.
authors.forename: Chungjung
authors.initials: C
authors.lastname: Chou
authors.affiliation: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Deiters
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D009636:Norbornanes / Q000737:chemistry*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D000596:Amino Acids; D005456:Fluorescent Dyes; D009636:Norbornanes; D011506:Proteins; D012235:Rhodamines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine