DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells.
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
Hui
H
Biotium, Inc., 3423 Investment Blvd., Ste. 8, Hayward, CA 94545, USA. dcen@biotium.com
Mao
Fei
F
Aronchik
Ida
I
Fuentes
Rholinelle Joy
RJ
Firestone
Gary L
GL
D001665:Binding Sites; D053148:Caspase 3 / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D019169:Jurkat Cells / Q000201:enzymology; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism*; D013379:Substrate Specificity
D010446:Peptide Fragments; D004247:DNA; D053148:Caspase 3