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Genetically targeted chromophore-assisted light inactivation

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Abstract

Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI)1 uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and α1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs2 that specifically bind the membrane-permeant, red biarsenical dye, ReAsH3,4. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by ∼90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or α1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.

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Figure 1: Staining and photoinactivation of TC-tagged gap junctions.
Figure 2: Comparing CALI using ReAsH, FlAsH and fluorescent proteins.
Figure 3: CALI of α1C Ca2+ channels.

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Acknowledgements

We wish to thank M.M. Falk for providing the Cx43-GFP construct, M.H. Ellisman for providing the Cx43-TC construct and Richard W. Tsien for critical reading of the manuscript. This work was supported by the Howard Hughes Medical Institute, Department of Energy contract DE-AC03-76SF00098 and National Institutes of Health grant NS27177 to R.Y.T. S.R.A. was supported in part by the National Institutes of Health grant PO1 DK54441 (to S.S. Taylor).

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  1. David A Zacharias

    Present address: Whitney Laboratory, 9505 Ocean Shore Blvd., St. Augustine, Florida, 32080, USA

Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093-0647, California, USA

    Oded Tour, David A Zacharias & Roger Y Tsien

  2. Dept. of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093-0647, California, USA

    Oded Tour, Rene M Meijer, David A Zacharias, Stephen R Adams & Roger Y Tsien

  3. Depts. of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093-0647, California, USA

    Roger Y Tsien

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  1. Oded Tour
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  2. Rene M Meijer
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Correspondence to Roger Y Tsien.

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Tour, O., Meijer, R., Zacharias, D. et al. Genetically targeted chromophore-assisted light inactivation. Nat Biotechnol 21, 1505–1508 (2003). https://doi.org/10.1038/nbt914

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