Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity.
Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity.
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DOI：
10.1371/journal.pone.0122513
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Jalink K
中科院分区：
文献类型：
--
作者：
Klarenbeek J;Goedhart J;van Batenburg A;Groenewald D;Jalink K
基于Epac的FRET传感器已被广泛用于检测活细胞中的cAMP浓度。最初由我们和其他人开发，从那时起，我们报告了几个重要的优化，使这些传感器受到许多细胞生物学家的喜爱。我们在这里报告克隆和表征我们的第四代cAMP传感器，其具有出色的光稳定性，动态范围和信噪比。该设计基于mTurquoise 2，目前最亮和最耐漂白的供体，以及一种新的受体盒，由两个cp173 Venus荧光团串联组成。我们还报告了在Epac部分具有单点突变Q270E的变体，其将cAMP的解离常数从9.5 μ M降低至4 μM，从而使亲和力增加约2.5倍。最后，我们还制备和表征了具有用于单波长FLIM采集的非发射（暗）受体的专用变体，其在cAMP水平饱和时显示出异常的近两倍的荧光寿命。我们相信这一代cAMP优于所有其他传感器，因此建议将这些传感器用于所有未来的研究。
Epac-based FRET sensors have been widely used for the detection of cAMP concentrations in living cells. Originally developed by us as well as others, we have since then reported several important optimizations that make these sensors favourite among many cell biologists. We here report cloning and characterization of our fourth generation of cAMP sensors, which feature outstanding photostability, dynamic range and signal-to-noise ratio. The design is based on mTurquoise2, currently the brightest and most bleaching-resistant donor, and a new acceptor cassette that consists of a tandem of two cp173Venus fluorophores. We also report variants with a single point mutation, Q270E, in the Epac moiety, which decreases the dissociation constant of cAMP from 9.5 to 4 μM, and thus increases the affinity ~ 2.5-fold. Finally, we also prepared and characterized dedicated variants with non-emitting (dark) acceptors for single-wavelength FLIM acquisition that display an exceptional near-doubling of fluorescence lifetime upon saturation of cAMP levels. We believe this generation of cAMP outperforms all other sensors and therefore recommend these sensors for all future studies.
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影响因子：
16.6
作者：
通讯作者：
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影响因子：
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作者：
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DOI：
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期刊：
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DOI：
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发表时间：
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影响因子：
11.1
作者：
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影响因子：
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作者：
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