Single-molecule visualization and manipulation of protein and DNA interactions and dynamics, on nanoscale dimensions

我的研究计划的首要目标是可视化、定量分析和了解蛋白质-DNA和蛋白质-蛋白质相互作用，它们在调节细胞过程中发挥着关键的生物物理作用。我对蛋白质和DNA分子等生物聚合物的动态、结构和遗传特性之间的相互作用很感兴趣，可视化和纳米操纵工具的进步，以及对受限分子的理论处理，使我们能够更详细地了解这些特性。这项发现资助将使我的团队能够利用我们的单分子显微镜工具来探索关于蛋白质和DNA系统在纳米尺度上的行为的公开问题；这些问题是其他方法无法获得的，并且具有国际研究兴趣和影响。*我的研究计划将与一个跨学科团队一起开展2个发现项目。项目1将可视化“超螺旋”DNA和“探测分子”之间的相互作用，这些分子被设计成结合到特定的“靶点”。这些研究将解决悬而未决的问题，即DNA的构象如何通过实时观察事件的发生来影响目标位点的解离，以及随后与这些位点的结合事件。这些测量将测试生物物理理论，这些理论正在被开发来描述DNA中广泛的“拓扑介导的过程”，其动机是试图了解活细胞中的基因调节。项目2将在纳米尺度上可视化蛋白质系统的行为；特别是我们将研究蛋白质-蛋白质相互作用如何调节“液滴”的形成，这反过来又在细胞内发挥特定的功能。这些项目将利用我们合作者在理论上的观点进行建模，在生物化学/分子生物学方面进行样品制备，并利用原子力显微镜进行高分辨率可视化。*我们独特的显微镜设备使我们能够在已知的限制和溶液条件下，通过轻轻地将单个分子“挤压”到定制的腔室中，获得分子相互作用和动力学的清晰电影。我们的技术使我们能够检测微弱和缓慢的相互作用事件，并在几十秒到几分钟内连续拍摄分子轨迹的电影。通过将分子限制在焦平面上，我们的方法是“免处理”的，允许分子在相互作用的同时探索其完整的构型空间；这意味着我们可以辨别DNA构象对蛋白质结合事件的影响。我们将设计分子系统，系统地增加复杂性，接近“细胞环境”中的条件。我们对生物物理机制的研究，通过新的物理工具与新的生物物理数据和建模相结合，将为生物医学的进步提供基础。

The overarching objective of my research program is to visualize, quantitatively analyze and understand protein-DNA and protein-protein interactions, which play key biophysical roles in regulating cellular processes. I am fascinated by the interplay between the dynamic, structural, and genetic properties of biopolymers such as protein and DNA molecules, which advancements in visualization and nano manipulation tools, as well as theoretical treatments of confined molecules, are allowing us to understand in further detail. This Discovery Grant will enable my group to leverage our single-molecule microscopy tools to explore open questions about the behavior of protein and DNA systems on nanoscale dimensions; questions which are inaccessible to other methods, and which have international research interest and impact.******My Research Program will pursue 2 Discovery Projects, with an interdisciplinary team. Project 1 will visualize the interactions between "supercoiled" DNA and "probe molecules" which are designed to bind to specific "target sites". These studies will address open questions about how the conformations of DNA can influence the unwinding of the target sites, as well as consequent binding events to these sites, by watching the events occur in real time. These measurements will test biophysical theories which are being developed to describe a wide range "topology-mediated processes" in DNA, motivated by seeking to understand gene regulation in living cells. Project 2 will visualize the behavior of protein systems on nanoscale dimensions; in particular we will study how protein-protein interactions regulate the formation of "liquid droplets", which in turn serve specific functionalities inside cells. These projects will leverage perspectives of our collaborators in theory for modeling, in biochemistry/molecular biology for sample preparation, and atomic force microscopy for high-resolution visualization.******Our unique microscopy devices enable crisp movies of molecular interactions and dynamics to be acquired, under known confinement and solution conditions, by gently "squeezing" individual molecules into custom chambers. Our techniques enable us to detect weak and slow interaction events and to take continuous movies of molecular trajectories over tens of seconds to minutes. By confining molecules to the focal plane, our approach is “handle-free”, allowing the molecules to explore their full configurational space while interacting with each other; this means we can discern the influence of DNA conformation upon protein-binding events, for example. We will design molecular systems to systematically increase in complexity, approaching conditions in the "cellular milieu". Our studies of biophysical mechanisms, enabled by new physical tools in combination with new biophysical data and modeling, will provide a foundation for advancements in biomedicine.