A Novel SFM-based Method for Studying Single Molecule Dynamics

描述(由申请人提供)：我们建议开发、分析、实施和测试一种在扫描力显微镜中跟踪单个粒子的新方法。研究单个分子的动力学和分子间相互作用的能力是分子生物学继续取得进展以及理解和治疗各种遗传病的关键组成部分。目前的技术包括在光学显微镜中使用单粒子跟踪和在光学陷阱中使用位置跟踪。光学显微镜中的粒子跟踪受到时间分辨率的限制。光学陷阱可以提供极高的空间和时间分辨率；然而，构建具有这种灵敏度的系统是极其具有挑战性的。由于扫描力显微镜具有精细的空间分辨率以及在液体中工作的能力，它已经成为研究单分子结构的标准工具。研究动力学的标准方法是使用时间推移成像。每幅图像可能需要几秒钟到几分钟的时间才能获取，因此这种方法的适用性极其有限。该方案的探索性研究集中于开发原子力显微镜的反馈控制算法，以直接跟踪单个分子，如马达蛋白质或聚合酶。我们的目标是(1)设计和测试算法，以在不成像的情况下沿着生物聚合物(如RNA、微管和肌动蛋白)快速移动末端，(2)将这些算法与检测和估计方案相结合，以跟踪在这些结构上运动的分子，例如分子马达或聚合酶，以及(3)应用该方案来研究酪蛋白肌球蛋白和肌球蛋白V沿肌动蛋白的运动。 与公共健康相关：在这个项目中，我们建议开发一种新的方法来研究单分子在生物聚合物上运动的动力学。该方法是一种以扫描力显微镜粒子跟踪概念为核心的新型控制方法。它利用了扫描力显微镜的高空间分辨率和悬臂梁的高共振频率所固有的高时间分辨率。

DESCRIPTION (provided by applicant): We propose to develop, analyze, implement and test a novel approach for tracking single particles in a scanning force microscope. The ability to study the dynamics of single molecules and of the interactions between molecules is a critical component for continued progress in molecular biology and for understanding and treating a variety of genetic diseases. Current techniques include using single particle tracking in optical microscopy and position tracking in optical traps. Particle tracking in optical microscopy is limited in its temporal resolution. Optical traps can provide superb spatial and temporal resolution; the construction of systems with such sensitivity, however, is extremely challenging. Due to the exquisite spatial resolution of scanning force microscopy as well as its ability to operate in liquid, it has become a standard tool for studying the structure of single molecules. The standard approach for studying dynamics is the use of time-lapse imaging. Each image can take seconds to minutes to acquire and thus the applicability of this approach is extremely limited. The exploratory research in this proposal is focused on developing feedback control algorithms for an atomic force microscope to directly track a single molecule such as a motor protein or a polymerase. We aim to (1) design and test algorithms for rapidly moving the tip along a biological polymer such as RNA, microtubules, and actin, without imaging, (2) combine these algorithms with detection and estimation schemes to track molecules moving on such structures, such as molecular motors or polymerases, and (3) apply the scheme to study the motion of tryopomyosin and of myosin V along actin. PUBLIC HEALTH RELEVANCE: In this project we propose to develop a novel method for studying the dynamics of single molecules moving on biopolymers. The method is a new control approach centered on the concept of particle tracking with a scanning force microscope. It takes advantage of the high spatial resolution of scanning force microscopy and the high temporal resolution inherent in the high resonant frequencies of the cantilevers.