Supercoiling DNA optically
Supercoiling DNA optically
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DOI：
10.1073/pnas.1908826116
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发表时间：
2019-12-26
影响因子：
11.1
通讯作者：
Wuite, Gijs J. L.
中科院分区：
文献类型：
--
作者：
King, Graeme A.;Burla, Federica;Wuite, Gijs J. L.
细胞DNA在基因组过程中经常受到扭转应力，如转录和复制，导致一系列超螺旋DNA结构。为此，在单分子水平上制备和研究超螺旋DNA的方法得到了广泛的应用，包括磁力、角光、微吸管和磁光镊子。然而，目前将DNA超螺旋控制与空间操纵和荧光显微镜相结合是具有挑战性的。这限制了研究超螺旋DNA复杂和动态相互作用的能力。在这里，我们提出了一种单分子分析，可以快速和可控地产生负的超螺旋DNA使用标准的双陷阱光镊仪。这种被称为光学DNA超螺旋(Ods)的方法，独一无二地结合了使用力光谱、整个DNA的荧光成像和快速缓冲交换来研究超螺旋DNA的能力。这项技术可以用来产生大范围的超卷曲状态，在
Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between