Booster, a Red-Shifted Genetically Encoded F?rster Resonance Energy Transfer (FRET) Biosensor Compatible with Cyan Fluorescent Protein/Yellow Fluorescent Protein-Based FRET Biosensors and Blue Light-Responsive Optogenetic Tools
Booster, a Red-Shifted Genetically Encoded F?rster Resonance Energy Transfer (FRET) Biosensor Compatible with Cyan Fluorescent Protein/Yellow Fluorescent Protein-Based FRET Biosensors and Blue Light-Responsive Optogenetic Tools
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Booster，一种红移基因编码 FRET 共振能量转移 (FRET) 生物传感器，与青色荧光蛋白/黄色荧光蛋白 FRET 生物传感器和蓝光响应光遗传学工具兼容
DOI：
10.1021/acssensors.9b01941
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发表时间：
2020
期刊：
影响因子：
8.9
通讯作者：
Matsuda Michiyuki
中科院分区：
文献类型：
--
作者：
Watabe Tetsuya;Terai Kenta;Sumiyama Kenta;Matsuda Michiyuki
基于基因编码的福斯特共振能量转移 (FRET) 的生物传感器已被开发用于信号分子活动的可视化。目前，它们大多数由青色和黄色荧光蛋白（CFP 和 YFP）组成，排除了在单个细胞内使用多个 FRET 生物传感器的可能性。此外，基于CFP和YFP的FRET生物传感器与在蓝光下操作的光遗传学工具不兼容。为了克服这些问题，我们开发了具有红移激发和发射波长的 FRET 生物传感器。通过计算福斯特距离，我们选择 mKOκ 和 mKate2 作为有利的供体和受体对。通过优化 FRET 生物传感器的荧光蛋白和调节域的顺序，我们开发了一种名为“Booster”的 FRET 生物传感器主干。基于 Booster 主链 (Booster-PKA) 的蛋白激酶 A (PKA) 生物传感器的性能与 AKAR3EV 相当，AKAR3EV 是之前开发的包含 CFP 和 YFP 的 FRET 生物传感器。为了验证概念，我们首先展示了使用基于 CFP 和 YFP 的 Booster-PKA 和 ERK FRET 生物传感器同时监测两种蛋白激酶的活性。其次，我们展示了 Beggiatoa 光激活腺苷酸环化酶（环 AMP 的光遗传学发生器）对 PKA 激活的监测。最后，我们展示了表达 Booster-PKA 的转基因小鼠活组织中的 PKA 活性。总的来说，结果证明了 Booster 生物传感器作为体外和体内成像工具的有效性和多功能性。
Genetically encoded Förster resonance energy transfer (FRET)-based biosensors have been developed for the visualization of signaling molecule activities. Currently, most of them are comprised of cyan and yellow fluorescent proteins (CFP and YFP), precluding the use of multiple FRET biosensors within a single cell. Moreover, the FRET biosensors based on CFP and YFP are incompatible with the optogenetic tools that operate at blue light. To overcome these problems, here, we have developed FRET biosensors with red-shifted excitation and emission wavelengths. We chose mKOκ and mKate2 as the favorable donor and acceptor pair by calculating the Förster distance. By optimizing the order of fluorescent proteins and modulatory domains of the FRET biosensors, we developed a FRET biosensor backbone named “Booster”. The performance of the protein kinase A (PKA) biosensor based on the Booster backbone (Booster-PKA) was comparable to that of AKAR3EV, a previously developed FRET biosensor comprising CFP and YFP. For the proof of concept, we first showed simultaneous monitoring of activities of two protein kinases with Booster-PKA and ERK FRET biosensors based on CFP and YFP. Second, we showed monitoring of PKA activation byBeggiatoaphotoactivated adenylyl cyclase, an optogenetic generator of cyclic AMP. Finally, we presented PKA activity in living tissues of transgenic mice expressing Booster-PKA. Collectively, the results demonstrate the effectiveness and versatility of Booster biosensors as an imaging tool in vitro and in vivo.