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A FRET-based probe with a chemically deactivatable quencher.

Geoffray Leriche¹, Ghyslain Budin, Zeinab Darwich

¹Laboratory of Functional Chemo-Systems UMR 7199, 74 Route du Rhin, 67401, Illkirch, France.

Chemical Communications (Cambridge, England)

|February 14, 2012

Summary

We developed a novel FRET-based probe with a chemically deactivatable quencher. This probe enables fluorescence turn-on via enzymatic cleavage or chemical reduction, allowing quantification of caspase-3 activity and detection of unreacted probes in cells.

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Area of Science:

Biochemistry
Chemical Biology
Molecular Probes

Background:

Förster Resonance Energy Transfer (FRET) probes are essential for monitoring biological processes.
Developing probes with tunable activation mechanisms is crucial for enhanced sensitivity and specificity.

Purpose of the Study:

To introduce a novel FRET-based probe concept utilizing a chemically deactivatable quencher.
To demonstrate the probe's ability to activate fluorescence through enzymatic cleavage or chemical reduction.
To validate the probe for quantifying caspase-3 activity and detecting unreacted probes in cellular environments.

Main Methods:

Design and synthesis of a FRET-based probe incorporating a chemically deactivatable quencher.
Enzymatic assays to measure caspase-3 cleavage activity.
Chemical reduction experiments using sodium dithionite to activate the probe.
Cell-based experiments to visualize unreacted probes.

Main Results:

The developed probe exhibits fluorescence turn-on upon caspase-3 enzymatic cleavage.
The probe's fluorescence can also be activated by the chemical reductant sodium dithionite.
The FRET probe successfully quantified caspase-3 cleavage activity in solution.
Unreacted probes were successfully visualized in cellular experiments.

Conclusions:

The chemically deactivatable quencher concept offers a versatile strategy for developing novel FRET-based probes.
This approach enables sensitive quantification of enzymatic activity and allows for the detection of probe distribution in complex biological systems.