Fluorogenic Rhodamine-Based Chemigenetic Biosensor for Monitoring Cellular NADPH Dynamics,Journal of the American Chemical Society

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Fluorogenic Rhodamine-Based Chemigenetic Biosensor for Monitoring Cellular NADPH Dynamics
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2024-07-22 , DOI: 10.1021/jacs.3c13137
Huimin Chang ₁ , Simon Clemens ₂ , Pingting Gao ₁ , Quanlin Li ₁ , Hanqing Zhao ₁ , Lehua Wang ₁ , Jingye Zhang ₁ , Pinghong Zhou ₁ , Kai Johnsson _{2,

3} , Lu Wang ₁

Affiliation

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.

中文翻译：

用于监测细胞 NADPH 动态的基于荧光罗丹明的化学遗传生物传感器

采用福斯特共振能量转移 (FRET) 的比率生物传感器能够实时跟踪代谢物动态。在这里，我们介绍了一种生成基于 FRET 的生物传感器的方法，其中表观 FRET 效率的变化依赖于罗丹明的分析物控制的荧光性，而不是常用的供体-受体荧光团之间的距离变化。我们的基于罗丹明的荧光化学遗传生物传感器 ( FOCS ) 依赖于合成的蛋白质束缚 FRET 探针，其中充当 FRET 受体的罗丹明以分析物依赖性方式从黑暗状态切换到荧光状态。这允许对分析物浓度进行比例感测。我们使用这种方法来生成烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的化学遗传学生物传感器。 FOCS-NADPH对 NAPDH 表现出快速、可逆的响应，具有良好的动态范围、选择性和 pH 不敏感性。 FOCS-NADPH可以实时监测氧化应激期间或药物暴露后活细胞中胞质 NADPH 的波动。我们还使用FOCS-NADPH通过葡萄糖代谢的磷酸戊糖途径研究 NADPH 稳态调节。 FOCS-NADPH是研究 NADPH 代谢的强大工具，并可作为未来荧光生物传感器开发的蓝图。

更新日期：2024-07-22

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