Direct Visualization of the Movement of a Single T7 RNA Polymerase and Transcription on a DNA Nanostructure
Direct Visualization of the Movement of a Single T7 RNA Polymerase and Transcription on a DNA Nanostructure
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DOI：
10.1002/anie.201201890
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Sugiyama, Hiroshi
中科院分区：
文献类型：
--
作者：
Endo, Masayuki;Tatsumi, Koichi;Sugiyama, Hiroshi
直接可视化完整的酶与DNA的相互作用是研究酶的机械行为和理解生物过程的最终方法之一。[1，2] RNA聚合酶（RNAP）转录是最重要的生物学过程之一。转录涉及一系列RNAP行为，包括与双链DNA（dsDNA）结合、沿dsDNA沿着滑动、RNA合成以及从dsDNA解离。RNA聚合酶在转录过程中的分子行为已经通过原子力显微镜（AFM）[3-6]和荧光显微镜进行了研究。[7-10]最近，高速AFM已经能够在真实的时间内以纳米级分辨率可视化单个分子的运动。[11-14]为了对单分子酶促反应进行一致的成像，需要一个观察平台。DNA折纸自组装系统能够将特定的DNA链放置在自组装支架上，可用于设计各种各样的纳米结构。[15-20]我们最近开发了一种这样的DNA支架，“DNA框架”，它将各种长度的dsDNA掺入支架的特定位置，并使单酶运动和反应可视化。[21-23]在这项研究中，我们使用基于AFM的单分子系统直接观察转录。我们使用DNA折纸方法制备了一个纳米级的观察平台（图1a）。将含有T7启动子的模板dsDNA（1000 bp）连接在该平台上的两个特定位置（图1b）。利用DNA折纸纳米结构
Direct visualization of intact enzymes interacting with DNA is one of the ultimate methods for investigating the mechanical behavior of enzymes and understanding biological processes.[1, 2] Transcription by RNA polymerase (RNAP) is one of the most important biological processes. Transcription involves a series of RNAP behaviors, including binding to double-stranded DNA (dsDNA), sliding along the dsDNA, RNA synthesis, and dissociation from the dsDNA. The molecular behavior of RNA polymerase during transcription has been investigated by atomic force microscopy (AFM)[3–6] and fluorescence microscopy.[7–10] Recently, high-speed AFM has enabled visualization the movements of single molecules at nanoscale resolution in real time.[11–14] For consistent imaging of single-molecule enzymatic reactions, an observation platform is required. The DNA origami self-assembly system, which enables specific DNA strands to be placed onto a self-assembled scaffold, can be used to design a wide variety of nanostructures.[15–20] We recently developed one such DNA scaffold, the “DNA frame”, which incorporates dsDNAs of various lengths into the scaffold at specific positions and enables visualization of single-enzyme movements and reactions.[21–23]In this study, we directly observed transcription using an AFM-based single-molecule system. We prepared a nanoscale observation platform using the DNA origami method (Figure1a). A template dsDNA (1000bp) containing the T7 promoter was attached at two specific positions on this platform (Figure 1b). Using the DNA origami nanostructure