Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China; Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China.
Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China; Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China.
Free Radic Biol Med. 2018 May 20;120:255-265. doi: 10.1016/j.freeradbiomed.2018.03.041. Epub 2018 Mar 23.
Genetically encoded fluorescent sensors are widely used to visualize secondary messengers, metabolites and dynamic events in living cells. However, almost all of these sensors are based on Aequorea GFPs or GFP-like proteins, which do not correctly maturate and fluoresce under hypoxia or anoxic conditions, greatly limiting their application in biomedical research. Herein, we provide a novel strategy for design of sensors and report a series of thiol redox-sensitive sensor based on a recently discovered oxygen-independent fluorescent protein UnaG from Japanese eel. These redox sensors have large dynamic range, rapid responsiveness, a flexible "switch", and pH-independence, are particularly compatible with hypoxia conditions, and therefore represent a substantial improvement for live-cell redox measurement. We further demonstrated the versatility of these redox sensors, by simultaneously monitoring redox changes and hypoxia state in living cells, thereby proving its capability as a powerful and flexible tool for indexing multidimensional metabolism data in the context of physiological stressors and pathological states. These redox sensors are not only the first case of UnaG-based functional sensors, but also the first case of functional sensors based on non GFP-like proteins. Based on this strategy, more oxygen-independent biosensors could be developed, hence, provide new opportunities for bioimaging.
遗传编码的荧光传感器被广泛用于可视化活细胞中的第二信使、代谢物和动态事件。然而，几乎所有这些传感器都是基于海肾 GFP 或 GFP 样蛋白，它们在缺氧或无氧条件下不能正确成熟和发光，这极大地限制了它们在生物医学研究中的应用。在此，我们提供了一种设计传感器的新策略，并报告了一系列基于最近从日本鳗鲡中发现的不依赖于氧的荧光蛋白 UnaG 的硫醇氧化还原敏感传感器。这些氧化还原传感器具有大的动态范围、快速响应性、灵活的“开关”和 pH 独立性，特别适合于低氧条件，因此代表了活细胞氧化还原测量的重大改进。我们进一步证明了这些氧化还原传感器的多功能性，通过同时监测活细胞中的氧化还原变化和低氧状态，从而证明其作为在生理应激和病理状态下索引多维代谢数据的强大和灵活工具的能力。这些氧化还原传感器不仅是基于 UnaG 的功能传感器的首例，也是基于非 GFP 样蛋白的功能传感器的首例。基于这种策略，可以开发更多的不依赖于氧的生物传感器，从而为生物成像提供新的机会。
