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Fluorogenic Rhodamine-Based Chemigenetic Biosensor for Monitoring Cellular NADPH Dynamics.
- Source :
-
Journal of the American Chemical Society [J Am Chem Soc] 2024 Jul 31; Vol. 146 (30), pp. 20569-20576. Date of Electronic Publication: 2024 Jul 22. - Publication Year :
- 2024
-
Abstract
- Ratiometric biosensors employing Förster Resonance Energy Transfer (FRET) enable the real-time tracking of metabolite dynamics. Here, we introduce an approach for generating a FRET-based biosensor in which changes in apparent FRET efficiency rely on the analyte-controlled fluorogenicity of a rhodamine rather than the commonly used distance change between donor-acceptor fluorophores. Our fluorogenic, rhodamine-based, chemigenetic biosensor ( FOCS ) relies on a synthetic, protein-tethered FRET probe, in which the rhodamine acting as the FRET acceptor switches in an analyte-dependent manner from a dark to a fluorescent state. This allows ratiometric sensing of the analyte concentration. We use this approach to generate a chemigenetic biosensor for nicotinamide adenine dinucleotide phosphate (NADPH). FOCS-NADPH exhibits a rapid and reversible response toward NAPDH with a good dynamic range, selectivity, and pH insensitivity. FOCS-NADPH allows real-time monitoring of cytosolic NADPH fluctuations in live cells during oxidative stress or after drug exposure. We furthermore used FOCS-NADPH to investigate NADPH homeostasis regulation through the pentose phosphate pathway of glucose metabolism. FOCS-NADPH is a powerful tool for studying NADPH metabolism and serves as a blueprint for the development of future fluorescent biosensors.
Details
- Language :
- English
- ISSN :
- 1520-5126
- Volume :
- 146
- Issue :
- 30
- Database :
- MEDLINE
- Journal :
- Journal of the American Chemical Society
- Publication Type :
- Academic Journal
- Accession number :
- 39037873
- Full Text :
- https://doi.org/10.1021/jacs.3c13137