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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/tRNACUA 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.

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Figure 1: Scheme to label proteins via an inverse electron-demand Diels–Alder cycloaddition, and structural formulae of relevant compounds.
Figure 2: Efficient, genetically-encoded incorporation of 2 using the PylRS/tRNACUA pair in E. coli.
Figure 3: Characterization of tetrazine–norbornene reactions.
Figure 4: Site-specific incorporation of 2 into proteins in mammalian cells and the specific labelling of EGFR-GFP on the cell surface with 9.

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Acknowledgements

We thank the Medical Research Council (U105181009, UD99999908), the European Research Council and the National Institutes for Health (GM079114) for funding. J.T.K. was supported by the National Science Foundation Graduate Research Fellowship under Grant No. NSF 0750733. We thank R. Mehl, who pointed out developments in inverse electron-demand Diels–Alder reactions while on sabbatical in the Chin lab.

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K.L, L.D. J.T.K., C.C., A.D. & J.W.C. designed the research and analysed the data. K.L, L.D., J.T.K. and C.C. performed the experiments. K.L. and J.W.C co-wrote the paper with input from the co-workers.

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Correspondence to Alexander Deiters or Jason W. Chin.

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Lang, K., Davis, L., Torres-Kolbus, J. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nature Chem 4, 298–304 (2012). https://doi.org/10.1038/nchem.1250

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