Caspase-3 (CPP32, apopain, YAMA), a member of asparate-specific cysteinyl proteases (or caspases) family, is a key mediator of apoptosis of mammalian cells [Kothakota et al., 1997]. Caspase-3 is activated during the early stages of apoptosis by self-proteolysis and/or cleavage by another protease. Active caspase-3 cleaves and activates caspases and many other cellular proteins, leading to apoptotic chromatin condensation and DNA fragmentation in all cell types examined [Porter and Janicke, 1999].

Casper3-GR is a FRET based sensor that can be used for detection of caspase-3 mediated apoptosis in living cells. The sensor consists of green and red fluorescent proteins TagGFP and TagRFP connected by the linker containing caspase-3 cleavage sequence DEVD. The high fluorescence quantum yield of TagGFP along with the high molar extinction coefficient of TagRFP and excellent overlap of donor emission and acceptor excitation spectra result in highly effective FRET between these fluorescent proteins [Shcherbo et al., 2009].

The activation of caspase-3 during apoptosis leads to cleavage of DEVD sequence and elimination of FRET that can be detected as decrease in the red emission of TagRFP and a simultaneous increase in green emission of TagGFP. Direct monitoring of the donor/acceptor emission ratio demonstrated up to 5-fold ratio changes upon cleavage by recombinant caspase 3 in vitro. The increase in donor fluorescence intensity was at least 2-fold corresponding to a FRET efficiency of at least 50%.