Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).
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This research was supported in part by the National Institutes of Health (1R01GM112728), the National Science Foundation (MCB-1330746) and the Charles E. Kaufman Foundation of The Pittsburgh Foundation. K.M. is grateful for a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad. We thank the Chin lab (Medical Research Council) for plasmids encoding the PylRS and PylT genes, the Asokan lab (University of North Carolina) for the pgRNA and pIRG plasmids and the Hughes lab (Montana State University) for the Cre Stoplight plasmid.
The authors declare no competing financial interests.
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Luo, J., Liu, Q., Morihiro, K. et al. Small-molecule control of protein function through Staudinger reduction. Nature Chem 8, 1027–1034 (2016). https://doi.org/10.1038/nchem.2573
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