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  • Homogeneous detection of caspase-3 using intrinsic fluorescence resonance energy transfer (iFRET).

Homogeneous detection of caspase-3 using intrinsic fluorescence resonance energy transfer (iFRET).

Biosensors & bioelectronics (2014-09-15)
Hyo Jin Kang, Ju Hwan Kim, Sang J Chung
ABSTRACT

Caspase-3 is an apoptotic cysteine protease and its aberrancy is highly implicated to numerous diseases thereby rendering caspase-3 activity as an important disease marker. Most caspase-3 sensors are caspase-3 substrates of which the fluorescence signals are turned on upon catalytic cleavage by active caspase-3. However, once the signal is generated, the fluorescence does not disappear albeit caspase-3 activity is abolished. Recently, we and other groups have developed the intrinsic Förster resonance energy transfer (iFRET) technique, which utilizes tryptophan residues of the target proteins and target-specific probes, as FRET donors and acceptors, respectively. Due to this principle, iFRET does not require the labeling of target proteins. In this work, we report the development of caspase-3 specific iFRET probes by structure-based design and synthesis, and the successful detection of caspase-3 in cell lysates as well as in its purified form. The limit-of-detection (LOD) of the probes in case of purified caspase-3 was found to be 1.4-1.5 nM. The designed probes did not bind to either procaspase-3 or C163S caspase-3, which are catalytic inactive, confirming that the observed iFRET signal correlates to the catalytic activity of caspase-3. Furthermore, in competition experiments with Ac-DEVD-CHO, a known competitive inhibitor of caspase-3, the iFRET signal was inhibited.

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