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Fluorogenic Rhodamine-Based Chemigenetic Biosensor for Monitoring Cellular NADPH Dynamics
- Source :
- Journal of the American Chemical Society; 20240101, Issue: Preprints
- 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-NADPHexhibits a rapid and reversible response toward NAPDH with a good dynamic range, selectivity, and pH insensitivity. FOCS-NADPHallows real-time monitoring of cytosolic NADPH fluctuations in live cells during oxidative stress or after drug exposure. We furthermore used FOCS-NADPHto investigate NADPH homeostasis regulation through the pentose phosphate pathway of glucose metabolism. FOCS-NADPHis a powerful tool for studying NADPH metabolism and serves as a blueprint for the development of future fluorescent biosensors.
Details
- Language :
- English
- ISSN :
- 00027863 and 15205126
- Issue :
- Preprints
- Database :
- Supplemental Index
- Journal :
- Journal of the American Chemical Society
- Publication Type :
- Periodical
- Accession number :
- ejs66964034
- Full Text :
- https://doi.org/10.1021/jacs.3c13137