A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors
Abstract
The control of cell death is an intricate process involving a multitude of intracellular modulators. Among these molecules, the caspases have a central role and have become an interesting group of enzymes in the current pharmaceutical industry. We have developed a novel dual-step fluorescence energy transfer-based separation-free assay method for the primary screening of caspase-3 inhibitors in vitro. This method relies on fluorescent europium(III)-chelate-doped nanoparticle donors coated with streptavidin in conjunction with a dual-labeled (N-terminal Alexa Fluor 680 fluorescent acceptor and C-terminal BlackBerry Quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety. In the assay, the nanoparticle donor excites the fluorescent acceptor, whose emission is monitored with time-resolved measurements. The intensity of the acceptor reflects the activity of the enzyme because the intensity is controlled by the proximity of the quencher. Owing to the dual-step fluorescence resonance energy transfer, this method enables a sensitized fluorescence signal directly proportional to the extent of enzymatic activity with relatively background fluorescence-free measurements in the event of complete enzyme inhibitio...Continue Reading
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