Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions
Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions
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DOI：
10.1039/c0cp02844d
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发表时间：
2011-01-01
影响因子：
3.3
通讯作者：
Peterman, Erwin J. G.
中科院分区：
文献类型：
--
作者：
Candelli, Andrea;Wuite, Gijs J. L.;Peterman, Erwin J. G.
复杂性和异质性是细胞内发生的许多分子事件的共同因素。单分子技术是量化生物分子作用的重要工具。然而，多个蛋白质之间的异质性相互作用很难用这些技术来研究。一种解决方案是将光学捕获与微流体和单分子荧光显微镜相结合。这种组合打开了研究异质/复杂蛋白质相互作用的可能性，具有前所未有的精确度和控制水平。它对于DNA-蛋白质相互作用的研究特别强大，因为它允许操纵DNA，同时可以可视化与其结合的单个蛋白质。在这项工作中，我们的目标是说明几个已发表和未发表的关键结果，采用荧光显微镜和光镊的组合。例如，最近对DNA和DNA-蛋白质复合物的结构特性、核蛋白丝在DNA上组装的分子机制以及DNA结合蛋白质的运动的研究。此外，我们提出了新的结果表明，单一的，荧光标记的蛋白质结合到个人，光学捕获的DNA分子已经可以跟踪与定位精度在10 nm以下的范围内，在1 pN以上的张力。我们和其他人的这些实验证明了这种单分子技术组合在复杂DNA-蛋白质相互作用研究中的巨大潜力。
Complexity and heterogeneity are common denominators of the many molecular events taking place inside the cell. Single-molecule techniques are important tools to quantify the actions of biomolecules. Heterogeneous interactions between multiple proteins, however, are difficult to study with these technologies. One solution is to integrate optical trapping with micro-fluidics and single-molecule fluorescence microscopy. This combination opens the possibility to study heterogeneous/complex protein interactions with unprecedented levels of precision and control. It is particularly powerful for the study of DNA-protein interactions as it allows manipulating the DNA while at the same time, individual proteins binding to it can be visualized. In this work, we aim to illustrate several published and unpublished key results employing the combination of fluorescence microscopy and optical tweezers. Examples are recent studies of the structural properties of DNA and DNA-protein complexes, the molecular mechanisms of nucleo-protein filament assembly on DNA and the motion of DNA-bound proteins. In addition, we present new results demonstrating that single, fluorescently labeled proteins bound to individual, optically trapped DNA molecules can already be tracked with localization accuracy in the sub-10 nm range at tensions above 1 pN. These experiments by us and others demonstrate the enormous potential of this combination of single-molecule techniques for the investigation of complex DNA-protein interactions.