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- Title
Genetically targeted chromophore-assisted light inactivation.
- Authors
Tour, Oded; Meijer, Rene M.; Zacharias, David A.; Tsien, Roger Y.; Adams, Stephen R.
- 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) 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 a1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. 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 a1C 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.
- Subjects
REACTIVE oxygen species; MICROINJECTIONS; CALCIUM channels; GREEN fluorescent protein; CONNEXINS; STAINS &; staining (Microscopy); THERAPEUTIC use of proteins
- Publication
Nature Biotechnology, 2003, Vol 21, Issue 12, p1505
- ISSN
1087-0156
- Publication type
Article
- DOI
10.1038/nbt914