Fluorescent biosensors enable to study cell physiology with spatiotemporal resolution, yet most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Forster Resonance Energy Transfer (FRET) pairs with near quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by either changing the fluorescent protein or the synthetic fluorophore, which enabled to monitor simultaneously free NAD+ in different subcellular compartments upon genotoxic stress. Minimal modifications furthermore allow the readout of these biosensors to be switched to fluorescence intensity, lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors.

中文翻译：

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开发具有大动态范围的多色生物传感器的通用方法

荧光生物传感器能够以时空分辨率研究细胞生理学，但大多数生物传感器的动态范围相对较低。在这里，我们介绍了一系列设计的 Forster 共振能量转移 (FRET) 对，它们基于荧光蛋白与荧光标记的 HaloTag 的可逆相互作用，具有接近定量的 FRET 效率。这些 FRET 对实现了钙、ATP 和 NAD+ 生物传感器的直接设计，具有前所未有的动态范围。这些生物传感器中的每一个的颜色都可以通过改变荧光蛋白或合成荧光团来轻松调整，从而能够在基因毒性应激时同时监测不同亚细胞区室中的游离 NAD+。此外，最小的修改允许将这些生物传感器的读数切换为荧光强度，寿命或生物发光。因此，这些 FRET 对为开发高度敏感和可调谐的生物传感器建立了一个新概念。