用于活细胞NADPH定量监测的半合成蛋白荧光探针

还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）作为生物体中普遍存在的重要辅酶和电子供体，具有调控细胞氧化应激和合成代谢反应等重要功能，而其代谢异常则与癌症等重大疾病的发生密切相关。然而，由于NADPH结构复杂且存在类似物，导致目前可用于NADPH实时定量监测的特异性荧光探针极其稀缺。本项目拟开发一种新的分子识别策略，结合蛋白响应型小分子染料与NADPH识别蛋白，构建半合成蛋白探针。小分子染料连有识别蛋白底物，而NADPH通过协同效应可增强底物和识别蛋白的结合，导致小分子染料靠近蛋白并相互作用，进而产生比率型荧光信号变化。通过转染表达蛋白和孵育可快速透膜的小分子染料，即可实现活细胞内NADPH可视化实时定量研究。这种全新分子识别策略不依赖于蛋白构象变化，设计简便且普适性高，利于高效开发特异性荧光探针，为复杂生物分子实时、动态、定量监测提供重要工具。

Reduced nicotinamide adenine dinucleotide phosphate (NADPH), as the key coenzyme and electron donor in biological systems, plays essential roles in cellular anabolic reactions and antioxidative effects. Meanwhile, NADPH metabolic disorder is highly correlated with many serious diseases, such as cancer. However, few fluorescent probes were developed and utilized for quantitatively imaging NADPH in living cells due to the complex structure of NADPH and the interference from its analogs. Here, we propose a new sensing strategy to produce semisynthetic sensor protein, which consists of a protein-responsive probe and an NADPH binding protein. The probe was conjugated with a substrate of the binding protein. NADPH can benefit the binding between substrate and protein through the synergistic effect, which brings the probe close to the protein. The interaction between probe and protein will cause the ratiometric fluorescence change. The incubation between the binding protein expressed inside cells via the transfection and the cell-permeable probe will allow the quantitive imaging of NADPH in living cells. The new design method, which is independent of the proteins’ conformation change, will offer a general strategy to create numerous fluorescent probes for real-time and quantitative imaging of biomolecules with complex structures.