title: Homogeneous detection of caspase-3 using intrinsic fluorescence resonance energy transfer (iFRET).
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.
authors.affiliation: Research Institute for Biomolecular Chemistry, Dongguk University, Seoul 100-715, Republic of Korea.
authors.forename: Hyo Jin
authors.initials: HJ
authors.lastname: Kang
authors.affiliation: Research Institute for Biomolecular Chemistry, Dongguk University, Seoul 100-715, Republic of Korea; NanoBio Major, Korea University of Science and Technology (UST), Daejeon 305-333, Republic of Korea.
authors.forename: Ju Hwan
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Research Institute for Biomolecular Chemistry, Dongguk University, Seoul 100-715, Republic of Korea; Department of Chemistry, Dongguk University, Seoul 100-715, Republic of Korea. Electronic address: sjchung@dongguk.edu.
authors.forename: Sang J
authors.initials: SJ
authors.lastname: Chung
mesh_terms: D015374:Biosensing Techniques; D053148:Caspase 3 / Q000737:chemistry / Q000302:isolation & purification*; D002384:Catalysis; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D009842:Oligopeptides / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D053148:Caspase 3
keywords: Caspase-3; Fluorescence sensor; Intrinsic fluorescence; Native protein detection; iFRET