Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic β-cells.

与基于绿色荧光蛋白的生物传感器相比，基于红色荧光蛋白（RFP）的生物传感器具有固有的优势，因为它具有降低的光毒性、减少的自发荧光和增强的组织穿透性。然而，现有的基于 RFP 的生物传感器常常存在动态范围小、定位错误和不需要的光转换等问题。此外，可用的基于 RFP 的生物传感器的选择是有限的，并且每种生物传感器的开发都需要大量的努力。在此，我们描述了一种通用且方便的方法，该方法将基因编码的非规范氨基酸 3-氨基酪氨酸引入绿色荧光蛋白样蛋白和生物传感器的发色团中，以实现自发且有效的绿色到红色的转换。我们证明了这种方法可用于快速扩展基于 RFP 的生物传感器的功能。经过很少的优化，3-氨基酪氨酸修饰的生物传感器保留了其绿色荧光前身的分子亮度、动态范围和响应能力。我们进一步应用光谱分辨生物传感器对胰腺 β 细胞代谢动力学进行多重成像。