Single-molecule pull-down for investigating protein-nucleic acid interactions.
Single-molecule pull-down for investigating protein-nucleic acid interactions.
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DOI：
10.1016/j.ymeth.2016.03.022
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发表时间：
2016-08-01
期刊：
影响因子：
--
通讯作者：
Joo C
中科院分区：
文献类型：
--
作者：
Fareh M;Loeff L;Szczepaniak M;Haagsma AC;Yeom KH;Joo C
基因组和转录组不断被细胞中的蛋白质修饰。单分子技术的最新进展允许对蛋白质和核酸之间的这些相互作用进行高空间和时间观察。然而，由于获得功能性蛋白质复合物的困难，研究大分子蛋白质复合物与核酸之间的相互作用仍然具有挑战性。在这里，我们结合单分子荧光与各种蛋白质复合物下拉技术，以确定核糖核蛋白复合物的功能和化学计量。通过使用来自真核细胞的蛋白质复合物的三个例子（Drosha，Dicer和TUT4蛋白质复合物），我们为使用新的单分子技术提供了逐步的指导。我们的单分子方法提供了亚秒级和纳米级的分辨率，可应用于细胞过程所必需的其他核蛋白复合物。
The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes.