Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.
Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.
复制标题
改善绿色和红色荧光蛋白的亮度和光稳定性，用于活细胞成像和FRET报告。
DOI：
10.1038/srep20889
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发表时间：
2016-02-16
影响因子：
4.6
通讯作者：
Chu J
中科院分区：
文献类型：
--
作者：
Bajar BT;Wang ES;Lam AJ;Kim BB;Jacobs CL;Howe ES;Davidson MW;Lin MZ;Chu J
许多基因编码的生物传感器使用福斯特共振能量转移（FRET）来动态报告生物分子的活动。虽然成对的青色和黄色荧光蛋白（FP）最常分别用作FRET伴侣荧光团，但绿色和红色FP为FRET提供了明显的优势，例如更大的光谱分离，更少的光毒性和更低的自发荧光。我们以前开发的绿色-红色FRET对三叶草和mRuby 2，提高了分子内FRET报告与不同的设计的响应性。在这里，我们报告了更明亮，更耐光的变体mClover 3和mRuby 3的工程。与上一代荧光团相比，mClover 3将光稳定性提高了60%，mRuby 3提高了200%。值得注意的是，mRuby 3也比mRuby 2亮35%，使其成为迄今为止最亮和最耐光的单体红色FP。此外，我们开发了一种标准化的方法，用于评估哺乳动物细胞中作为独立标记物和FRET伴侣的FP性能。我们发现mClover 3或mRuby 3在哺乳动物细胞中的表达分别提供了所有水母GFP或珊瑚RFP衍生物中最高的荧光信号。最后，使用mClover 3和mRuby 3，我们设计了具有更大响应幅度的CaMKIIα报告基因Camuiα的改进版本。
Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.