1] Department of Applied Physics, Stanford University, Stanford, California, USA. [2] Department of Bioengineering, Stanford University, Stanford, California, USA.
1] Department of Applied Physics, Stanford University, Stanford, California, USA. [2] Department of Bioengineering, Stanford University, Stanford, California, USA. [3].
Nat Methods. 2014 Apr;11(4):456-62. doi: 10.1038/nmeth.2854. Epub 2014 Feb 23.
Single-molecule measurements of DNA twist and extension have been used to reveal physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with high rotational drag, which prevents detection of short-lived intermediates or small angular steps. We introduce gold rotor bead tracking (AuRBT), which yields >100× improvement in time resolution over previous techniques. AuRBT employs gold nanoparticles as bright low-drag rotational and extensional probes, which are monitored by instrumentation that combines magnetic tweezers with objective-side evanescent darkfield microscopy. Our analysis of high-speed structural dynamics of DNA gyrase using AuRBT revealed an unanticipated transient intermediate. AuRBT also enables direct measurements of DNA torque with >50× shorter integration times than previous techniques; we demonstrated high-resolution torque spectroscopy by mapping the conformational landscape of a Z-forming DNA sequence.
单分子 DNA 扭转和延伸测量已被用于揭示双螺旋的物理性质，并用于研究核蛋白复合物中的结构动力学和机械化学。然而，由于使用具有高旋转阻力的角度探针，扭转测量的时空分辨率受到限制，这阻止了对短寿命中间体或小角度步骤的检测。我们引入了金转子珠跟踪（AuRBT），与以前的技术相比，该技术的时间分辨率提高了> 100 倍。AuRBT 使用金纳米颗粒作为明亮的低阻力旋转和延伸探针，通过将磁镊与目标侧消逝场暗场显微镜相结合的仪器进行监测。我们使用 AuRBT 对 DNA 拓扑异构酶的高速结构动力学进行的分析揭示了一种意想不到的瞬态中间体。AuRBT 还可以直接测量 DNA 扭矩，与以前的技术相比，积分时间缩短了> 50 倍；我们通过绘制 Z 形成 DNA 序列的构象景观，展示了高分辨率的扭矩光谱学。
Zotero 插件
