Nature Biotechnology
21, 1505 - 1508 (2003)
Published online: 16 November 2003; | doi:10.1038/nbt914
Genetically targeted chromophore-assisted light inactivationOded Tour1, 2, Rene M Meijer2, David A Zacharias1, 2, 4, Stephen R Adams2
& Roger Y Tsien1, 2, 31
Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA. 2
Dept. of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA. 3
Depts. of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA. 4
Present address: Whitney Laboratory, 9505 Ocean Shore Blvd., St. Augustine, Florida 32080, USA.
Correspondence should be addressed to Roger Y Tsien rtsien@ucsd.eduStudies 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  1C 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 1C 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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