Fluorescent proteins enable targeted visualization of biomolecules in living cells, but their maturation is oxygen-dependent and they are susceptible to aggregation and/or suffer from poor photophysical properties. Organic fluorophores are oxygen-independent with superior photophysical properties, but targeting biomolecules in vivo is challenging. Here, we introduce two oxygen-independent chemogenetic protein (OICP) tags that impart fluorogenicity and fluorescence lifetime enhancement to bound organic dyes. We present a photo- and physicochemical characterization of thirty fluorophores interacting with two OICPs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of the proteins. The binding specificity of the tags and the superior photophysical properties of organic fluorophores enable microscopy of living bacterial and eukaryotic cells. The exchange of photobleached dye for unbleached fluorophore enables prolonged live-cell imaging. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and microscopy applications under strictly oxygen-free conditions.

中文翻译：

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用于活细胞荧光显微镜的不依赖氧的化学生成蛋白标签

荧光蛋白能够使活细胞中的生物分子有针对性地显现，但是它们的成熟取决于氧气，它们易于聚集和/或遭受光物理性质的影响。有机荧光团不依赖氧，具有出色的光物理特性，但是在体内靶向生物分子具有挑战性。在这里，我们介绍了两个不依赖氧的化学生成蛋白（OICP）标签，这些标签赋予结合的有机染料荧光性和荧光寿命。我们提出了与两个OICP相互作用的三十个荧光团的光化学和物理化学特征，并得出结论，芳香族平面结构以高特异性结合到蛋白质的疏水口袋上。标签的结合特异性和有机荧光团的优异光物理特性使得能够对活细菌和真核细胞进行显微镜检查。用光漂白染料交换未漂白的荧光团可以延长活细胞成像的时间。我们的蛋白质标签为在严格无氧的条件下研究（亚）细胞蛋白质定位和动力学，蛋白质-蛋白质相互作用以及显微镜应用提供了一种通用工具。