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颗粒的新方法。研究单分子动力学和分子间相互作用的能力是分子生物学持续进步以及理解和治疗各种遗传疾病的关键组成部分。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在其时间分辨率方面受到限制。光学陷阱可以提供极好的空间和时间分辨率;然而，具有这种灵敏度的系统的构建是极具挑战性的。由于扫描力显微镜的精密空间分辨率以及其在液体中操作的能力，它已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延时成像。每个图像可能需要几秒钟到几分钟的时间来获取，因此这种方法的适用性非常有限。该提案中的探索性研究集中在为原子力显微镜开发反馈控制算法，以直接跟踪单个分子，如运动蛋白或聚合酶。我们的目标是（1）设计和测试算法，用于快速移动的尖端沿着一个生物聚合物，如RNA，微管，和肌动蛋白，没有成像，（2）联合收割机这些算法与检测和估计计划，以跟踪分子移动的结构，如分子马达或聚合酶，和（3）应用该计划研究运动的tryopomyosin和肌球蛋白V沿着肌动蛋白。 公共卫生相关性：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移动的动力学。该方法是一种新的控制方法的概念为中心的粒子跟踪与扫描力显微镜。它利用了扫描力显微镜的高空间分辨率和高时间分辨率固有的高谐振频率的杠杆。

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.