Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, NY 10032, USA .
Biochem Biophys Res Commun. 2012 Oct 5;426(4):565-70. doi: 10.1016/j.bbrc.2012.08.127. Epub 2012 Sep 4.
We report a new approach to probing DNA-protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin-streptavidin linkage withstands ∼20pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ⩾65pN; access to this higher force regime is sufficient to probe the responses of protein-DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.
我们报告了一种新的方法来探测 DNA-蛋白质相互作用，将光学镊子与高通量 DNA 窗帘技术相结合。在这里，我们确定了从双层膜中去除单个脂质锚定 DNA 分子所需的力。我们证明，通过单个生物素-链霉亲和素连接锚定在双层膜上的 DNA 在被从双层膜中拉出来之前可以承受约 20pN 的力，而通过多个附着点锚定在双层膜上的分子可以承受 ⩾65pN 的力；进入这个更高的力区足以探测蛋白质-DNA 相互作用对力变化的响应。作为一个原理验证，我们同时可视化了 DNA 结合的荧光标记的 RNA 聚合酶，同时拉伸 DNA 分子。这项工作朝着一个强大的实验平台迈出了一步，该平台将能够在应用光学镊子施加定义力的同时同时可视化 DNA 窗帘。
