A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.
A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.
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DOI：
10.1371/journal.pone.0001916
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发表时间：
2008-04-02
期刊：
影响因子：
3.7
通讯作者：
Jalink, Kees
中科院分区：
文献类型：
--
作者：
van der Krogt, Gerard N. M.;Ogink, Janneke;Ponsioen, Bas;Jalink, Kees
我们最近报道了CFP-Epac-YFP，这是一种基于epac的单多肽FRET报告基因，用于检测活细胞中的cAMP水平。在本研究中，我们比较和优化了用于生物传感器（如CFP-Epac-YFP）的荧光蛋白供体/受体对。我们的策略是制备由不同的供体和受体荧光蛋白组成的广泛结构，这些荧光蛋白由短连接体分开。构建体在HEK293细胞中表达，并检测FRET和其他相关特性。最有希望的对随后被用于尝试改善基于epac的cAMP传感器的FRET跨度。结果表明，尽管不是完全相关，但间隔结构和Epac传感器的性能之间存在显著的相关性。最后，该策略使我们能够通过敏化发射和荧光寿命成像来识别改进的传感器。本综述将有助于指导未来FRET传感器的发展。
We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.
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DOI：
10.1073/pnas.97.22.11984
发表时间：
2000-10-24
影响因子：
11.1
作者：
Baird, GS;Zacharias, DA;Tsien, RY
通讯作者：
Tsien, RY
影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
影响因子：
4.8
作者：
He, YY;Huang, JL;Chignell, CF
通讯作者：
Chignell, CF
影响因子：
2.9
作者：
Patterson, GH;Piston, DW;Barisas, BG
通讯作者：
Barisas, BG
影响因子：
3.4
作者：
Peter, M;Ameer-Beg, SM;Ng, T
通讯作者：
Ng, T