Booster, a Red-Shifted Genetically Encoded Förster 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 Förster 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 是一种红移基因编码福斯特共振能量转移 (FRET) 生物传感器，与基于青色荧光蛋白/黄色荧光蛋白的 FRET 生物传感器和蓝光响应光遗传学工具兼容。
作者：
Tetsuya Watabe;Kenta Terai;K. Sumiyama;M. Matsuda
基于遗传编码的Förster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，它们中的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。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，在这里，我们已经开发出具有红移激发和发射波长的FRET生物传感器。通过计算Förster距离，我们选择mKOκ和mKate 2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控结构域的顺序，我们开发了一个FRET生物传感器骨架命名为“助推器”。基于Booster骨架（Booster-PKA）的蛋白激酶A（PKA）生物传感器的性能与先前开发的包含CFP和YFP的FRET生物传感器AKAR 3EV的性能相当。为了验证概念，我们首先展示了基于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。第二，我们显示了通过Beggiatoa光活化腺苷酸环化酶（一种环AMP的光遗传发生器）监测PKA活化。最后，我们提出了PKA活性的转基因小鼠表达Booster-PKA的活组织。总的来说，这些结果证明了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 by Beggiatoa photoactivated 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.
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影响因子：
17.3
作者：
Shcherbakova DM;Stepanenko OV;Turoverov KK;Verkhusha VV
通讯作者：
Verkhusha VV
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
2.9
作者：
Lillo, MP;Beechem, JM;Mas, MT
通讯作者：
Mas, MT
DOI：
10.1042/bj20081949
发表时间：
2009-03-15
期刊：
The Biochemical journal
影响因子：
--
作者：
Shcherbo D;Murphy CS;Ermakova GV;Solovieva EA;Chepurnykh TV;Shcheglov AS;Verkhusha VV;Pletnev VZ;Hazelwood KL;Roche PM;Lukyanov S;Zaraisky AG;Davidson MW;Chudakov DM
通讯作者：
Chudakov DM
影响因子：
3.4
作者：
Grant, David M.;Zhang, Wei;French, Paul M. W.
通讯作者：
French, Paul M. W.