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Updated: Mar 18, 2026
Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules
Published on: November 2, 2009
Development of ratiometric sirtuin fluorescence probes via cleavage of a coumarin dye
Mitsuyasu Kawaguchi1, Ushio Komoda1, Naoya Ieda1,2
1Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.
Abstract:
Dysfunction of sirtuins (SIRTs), a family of NAD+-dependent histone deacetylases involved in epigenetic modulation of protein function and gene expression, is associated with both age-related metabolic diseases and cancers in mammals, and SIRT modulators are considered attractive therapeutic targets. However, although various SIRT detection methodologies have been developed, especially focusing on other enzymatic activities of SIRTs, such as long chain defatty-acylase activity, there has been little progress in developing methodologies capable of quantitatively evaluating SIRT activity. Herein, we present the first Förster resonance energy transfer (FRET)-based ratiometric fluorescence probes for SIRT1, containing fluorescein isothiocyanate (FITC) as a FRET acceptor, coumarin as a FRET donor and an H3K9 SIRT recognition peptide sequence. Cleavage by SIRT of firstly designed probe released the coumarin fluorophore and structural modification of the latter units yielded compounds with high SIRT1 selectivity and reactivity. We confirmed that these probes enabled the quantitative measurement of SIRT1 enzyme activity. They are expected to be useful tools in studies of epigenetic regulation mechanisms, and in high-throughput screening for drug discovery targeting SIRT.
Insights
Researchers developed novel FRET-based probes to quantitatively measure SIRT1 enzyme activity. These tools aid epigenetic studies and drug discovery for metabolic diseases and cancers.
Area of Science:
- Biochemistry
- Epigenetics
- Molecular Biology
Background:
- Sirtuins (SIRTs) are NAD+-dependent enzymes crucial for epigenetic regulation.
- SIRT dysfunction is linked to metabolic diseases and cancers, making them therapeutic targets.
- Existing methods for detecting SIRT activity are limited, especially for quantitative measurements.
Purpose of the Study:
- To develop novel, quantitative fluorescence probes for SIRT1 activity.
- To create tools for advancing epigenetic research and drug discovery.
Main Methods:
- Designed Förster resonance energy transfer (FRET)-based ratiometric fluorescence probes incorporating FITC, coumarin, and an H3K9 peptide.
- Utilized SIRT-mediated cleavage of the probe to release a coumarin fluorophore.
- Optimized probe structure for enhanced SIRT1 selectivity and reactivity.
Main Results:
- Successfully developed the first FRET-based ratiometric probes for quantitative SIRT1 activity measurement.
- Demonstrated high selectivity and reactivity of the modified probes for SIRT1.
- Confirmed the probes' ability to accurately quantify enzyme activity.
Conclusions:
- The developed FRET probes offer a novel method for quantitative SIRT1 activity assessment.
- These probes are valuable tools for epigenetic mechanism studies.
- The probes are expected to facilitate high-throughput screening for SIRT1-targeting drug discovery.
