Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching.
Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching.
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DOI：
10.1039/c4ib00251b
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发表时间：
2015-02
期刊：
影响因子：
--
通讯作者：
Palmer AE
中科院分区：
文献类型：
--
作者：
Dean KM;Lubbeck JL;Davis LM;Regmi CK;Chapagain PP;Gerstman BS;Jimenez R;Palmer AE
荧光蛋白在活细胞和生物体中提供特殊的标记特异性。不幸的是，它们的生物物理性质对于低丰度细胞成分的长期成像仍然远远不够理想，这在很大程度上是因为它们的光稳定性差。尽管广泛的工程努力，提高荧光蛋白的光稳定性仍然具有挑战性，由于缺乏适当的高通量选择方法。在这里，我们使用分子动力学引导的诱变结合最近开发的基于微流体的平台，该平台根据细胞的荧光光稳定性对细胞进行分类，以识别来自HeLa细胞库的光漂白减少的红色荧光蛋白。确定的突变体，命名为Kriek，具有2.5倍和4倍的光稳定性比其祖先，mCherry，在宽场和共聚焦照明下，分别。此外，这些结果提供了对增强光稳定性的机制及其与其他生物物理过程的联系的深入了解，从而为正在进行的具有改进的单分子和低拷贝成像能力的荧光蛋白的开发提供了方向。荧光蛋白能够在整个可见光谱中原位成像，具有极好的分子特异性和单分子灵敏度。不幸的是，当与领先的小分子荧光团（例如，Cy3），荧光蛋白，遭受加速的光漂白和差的积分光子输出。这是由于缺乏适当的高通量方法来提高荧光蛋白的光稳定性，以及对荧光蛋白光漂白的分子理解不足。在这里，我们报告的第一个应用程序，最近开发的微流控细胞分选仪，以确定荧光蛋白从mCherry衍生的库具有改善的光稳定性。这些结果提供了对荧光蛋白生物物理学的深入了解，大大加快了改进突变体的鉴定，并且可以应用于遗传编码和小分子荧光团。
Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities. Fluorescent proteins enable imaging in situ, throughout the visible spectrum, with superb molecular specificity and single-molecule sensitivity. Unfortunately, when compared to leading small-molecule fluorophores (e.g., Cy3), fluorescent proteins, suffer from accelerated photobleaching and poor integrated photon output. This results from a lack of appropriate high-throughput methods for improving the photostability of fluorescent proteins, as well as a poor molecular understanding of fluorescent protein photobleaching. Here, we report the first application of a recently developed microfluidic cell-sorter to identify fluorescent proteins from a mCherry-derived library with improved photostability. The results provide insight into fluorescent protein photophysics, greatly accelerate identification of improved mutants, and can be applied to both genetically encoded and small-molecule fluorophores.
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影响因子：
3.7
作者：
Alieva NO;Konzen KA;Field SF;Meleshkevitch EA;Hunt ME;Beltran-Ramirez V;Miller DJ;Wiedenmann J;Salih A;Matz MV
通讯作者：
Matz MV
影响因子：
16.6
作者：
通讯作者：
--
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
DOI：
10.1073/pnas.1013910107
发表时间：
2010-11-23
影响因子：
11.1
作者：
Chica, Roberto A.;Moore, Matthew M.;Mayo, Stephen L.
通讯作者：
Mayo, Stephen L.
影响因子：
3.4
作者：
Harms, GS;Cognet, L;Schmidt, T
通讯作者：
Schmidt, T