Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA-Protein Interactions at the Single-Molecule Level.
Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA-Protein Interactions at the Single-Molecule Level.
复制标题
利用酵母中表达的生物素化蛋白质在单分子水平上可视化 DNA-蛋白质相互作用
DOI：
10.3389/fmicb.2017.02062
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发表时间：
2017
影响因子：
5.2
通讯作者：
Fu YV
中科院分区：
文献类型：
--
作者：
Xue H;Bei Y;Zhan Z;Chen X;Xu X;Fu YV
我们在传统生物化学方面的许多知识都来自于批量化验。然而，当在系综上求平均时，许多随机过程和瞬时中间产物是隐藏的。单分子荧光显微镜的强大技术为理解传统方法无法理解的生命过程做出了巨大贡献。在单分子研究中，量子点与其他荧光探针相比具有几个独特的优势，如高亮度、极高的光稳定性和大的斯托克斯位移，从而允许长期观察和改善信噪比。然而，到目前为止，还没有一种简便的方法来标记从萌芽酵母中提纯的蛋白质。基于Bira-Avi和生物素-链霉亲和素系统，我们建立了一种简单的方法来获得QDot标记的蛋白质，并用全内反射荧光显微镜观察其与DNA的相互作用。在概念验证方面，我们选择复制蛋白A(RPA)和起源识别复合体(ORC)作为感兴趣的蛋白质。从生物素化效率较高的芽殖酵母中提纯蛋白质，并用链霉亲和素包被的Qdots快速标记。成功地在单分子水平上观察到蛋白质与DNA之间的相互作用。
Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots) have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA) and origin recognition complex (ORC) as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.
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影响因子：
3.3
作者：
Bell SP;Labib K
通讯作者：
Labib K
DOI：
10.1016/j.ymeth.2016.03.022
发表时间：
2016-08-01
期刊：
Methods (San Diego, Calif.)
影响因子：
--
作者：
Fareh M;Loeff L;Szczepaniak M;Haagsma AC;Yeom KH;Joo C
通讯作者：
Joo C
DOI：
10.1073/pnas.1332608100
发表时间：
2003-06-24
影响因子：
11.1
作者：
de Boer, E;Rodriguez, P;Strouboulis, J
通讯作者：
Strouboulis, J
影响因子：
16.6
作者：
Burgers PMJ;Kunkel TA
通讯作者：
Kunkel TA
DOI：
10.1126/science.aan0063
发表时间：
2017-07-21
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Coster G;Diffley JFX
通讯作者：
Diffley JFX