Chang, HuiminClemens, SimonGao, Pingtingchen, yanlingZhao, HanqingWang, LehuaZhang, JingyeZhou, Pinghongjohnsson, kaiwang, lu2025-01-302025-01-302025-01-302024-07-2210.1021/jacs.3c13137https://infoscience.epfl.ch/handle/20.500.14299/245947WOS:00127450030000139037873Ratiometric biosensors employing Forster 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.EnglishFLUORESCENT-PROBESSNAP-TAGGLUCOSE-6-PHOSPHATE-DEHYDROGENASESENSORSScience & TechnologyPhysical Sciencestext::journal::journal article::research article