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Journals The FASEB Journal Volume 22 Issue 7

DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells Hui Cen * ,1 , Fei Mao * , Ida Aronchik † , Rholinelle Joy Fuentes * and Gary L. Firestone † * Biotium, Inc., Hayward, California, USA; and † Department of Molecular and Cell Biology, University of California, Berkeley, California, USA 1 Correspondence: Biotium, Inc., 3423 Investment Blvd., Ste. 8, Hayward, CA 94545, USA. E-mail: dcen@biotium.com Live-cell detection of intracellular enzyme activity requires that substrates are cell-permeable and that the generated products are easily detected and retained in cells. Our objective was to create a novel fluorogenic substrate that could be used for real-time detection of apoptosis in living cells. We have synthesized a highly cell-permeable caspase-3 substrate, DEVD-NucView488, by linking a fluorogenic DNA-binding dye to the caspase-3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD-NucView488 detected caspase-3 activation within a live-cell population much earlier and with higher sensitivity compared with other apoptosis reagents that are currently available. Furthermore, cells incubated with DEVD-NucView488 exhibited no toxicity and normal apoptotic progression. DEVD-NucView488 is an ideal substrate for kinetic studies of caspase-3 activation because it detects caspase-3 activity in real-time and also efficiently labels DNA in nuclei of caspase-3-activated cells for real-time fluorescent visualization of apoptotic morphology. The strategy utilized in the design of this fluorogenic substrate can be applied in future endeavors to develop substrates for detecting real-time intracellular enzyme activity.—Cen, H., Mao, F., Aronchik, I., Fuentes, R. J., Firestone, G. L. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. Key Words: apoptosis • fluorescence • imaging • nucleic acid • nucleus

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DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells

Cen, Hui; Mao, Fei; Aronchik, Ida; Fuentes, Rholinelle Joy; Firestone, Gary L.
The FASEB Journal , Volume 22 (7): 2243
Fed of American Socs for Experimental Biology – Jul 1, 2008

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