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

纳米尺度上蛋白质和 DNA 相互作用和动力学的单分子可视化和操纵

• 批准号: RGPIN-2017-04348
• 负责人: Leslie, Sabrina
• 金额: $ 3.35万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710973
• 关键词: Single; molecule; visualization; manipulation; protein

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.

我的研究计划的总体目标是可视化，定量分析和理解蛋白质-DNA和蛋白质-蛋白质相互作用，在调节细胞过程中发挥关键的生物物理作用。我着迷于生物聚合物（如蛋白质和DNA分子）的动态，结构和遗传特性之间的相互作用，可视化和纳米操作工具的进步，以及对受限分子的理论处理，使我们能够更详细地了解。这项发现补助金将使我的团队能够利用我们的单分子显微镜工具来探索有关纳米尺度上蛋白质和DNA系统行为的开放问题;其他方法无法访问的问题，以及具有国际研究兴趣和影响的问题。 我的研究计划将追求2发现项目，与跨学科的团队。项目1将可视化“超螺旋”DNA和“探针分子”之间的相互作用，这些分子被设计为与特定的“靶位点”结合。这些研究将通过观察真实的时间内发生的事件来解决有关DNA构象如何影响靶位点解旋以及随后与这些位点结合的事件的开放性问题。这些测量将测试生物物理理论，这些理论正在开发中，以描述DNA中广泛的“拓扑介导的过程”，其动机是寻求了解活细胞中的基因调控。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，从而在细胞内发挥特定的功能。这些项目将利用我们的合作者在理论建模，生物化学/分子生物学样品制备和原子力显微镜高分辨率可视化方面的观点。 我们独特的显微镜设备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到微弱和缓慢的相互作用事件，并在几十秒到几分钟内拍摄分子轨迹的连续电影。通过将分子限制在焦平面上，我们的方法是“无干扰的”，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，以系统地增加复杂性，接近“细胞环境”中的条件。我们对生物物理机制的研究，通过新的物理工具结合新的生物物理数据和建模，将为生物医学的进步提供基础。