Single-molecule dynamics in solution with anti-Brownian trapping

具有反布朗捕获的溶液中的单分子动力学

• 批准号: 10697872
• 负责人: Quan Wang
• 金额: $ 223.84万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697872
• 关键词: Algorithms; Area; Binding; Biophysics; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Data; Data Analyses; Detection; Development; Dissection; Dyes; Fluorescence Resonance Energy Transfer; Goals; Guide RNA; Heterogeneity; Holoenzymes; Hybrids; Individual; Joints; Light; Location; Measurement; Methods; Modality; Modeling; Molecular Conformation; Monitor; Nature; Noise; Pathway interactions; Positioning Attribute; Process; RNA; RNA Conformation; Reaction; Resolution; Series; Spectrum Analysis; Structural Models; Structure; Testing; Time; Universities; Work; analysis pipeline; computerized data processing; design; insight; interest; novel; single molecule; single-molecule FRET; statistics; tool

We have made progress on the following two areas during the past year a) Joint change-point detection of multiple measurement channels. During my previous position as an independent fellow at Princeton University, my lab have developed a new experimental platform called ABEL-FRET (Wilson and Wang, Nat. Methods 18, 816), which allows FRET dynamics of individual molecules to be monitored without tethering in solution. There are two major advantages of ABEL-FRET compared to conventional modalities: ultrahigh resolution of structural heterogeneities and simultaneous measurement of hydrodynamic parameters. Meanwhile, ABEL-FRET generates single-molecule time traces that are multidimensional in nature and poses new challenges for data analysis. In particular, a task of interest is to identify the locations of abrupt change points in the presence of measurement noise. These change points might occur in one measurement channel or in many channels in a correlated way. We developed a new analysis pipeline called MULLR (which stands for MUlti-channel Log-Likelihood Ratio test) to perform joint change point identification across multiple measurement channels. Being one of the first algorithms applicable to multichannel single-molecule time trace, MULLR is specifically designed to be model-free and can handle channels with different measurement statistics. We demonstrated MULLR on both simulated and experimental data and compared MULLR to alternative data processing strategies. This work further enhances ABEL-FRET to sense biomolecular processes at the single-molecule level. b) Single-molecule dissection of gRNA conformation during Cas9 holoenzyme assembly. Biomolecules carry out their function by cycling through a series of functional states. To better understand the structural-functional relations, it is of tremendous interest to probe structure at sequential stages of the functional states. We recently used the ABEL-FRET platform to probe the 3-end structure of CRISPR RNA (crRNA) at the single-molecule level as it assembles into the Cas9 holoenzyme. For every molecule, its assembly state is unambiguously determined using hydrodynamic profiling and its 3-end structure is probed by a pair of strategically placed FRET dyes. Strikingly, we discovered structural heterogeneity and dynamics at every stage of the assembly pathway that is, crRNA, guide RNA (gRNA, or crRNA-tracrRNA hybrid), Cas9-gRNA complex and Cas9-gRNA bound with substrate DNA, highlighting the importance of RNA structural diversity. Current work focuses on using RNA structural prediction tools to generate structural models consistent with single-molecule FRET measurements and devising plausible pathways of Cas9 holoenzyme assembly. This work could potentially shed light on fundamental biophysical principles of Cas9-gRNA recognition

过去一年我们在以下两个方面取得了进展 a) 多个测量通道的联合变点检测。在我之前担任普林斯顿大学独立研究员期间，我的实验室开发了一个名为 ABEL-FRET（Wilson 和 Wang，Nat.Methods 18, 816）的新实验平台，该平台允许监测单个分子的 FRET 动力学，而无需束缚在溶液中。与传统方式相比，ABEL-FRET 有两大优势：超高分辨率的结构异质性和同时测量流体动力学参数。同时，ABEL-FRET 生成的单分子时间轨迹本质上是多维的，给数据分析带来了新的挑战。特别是，感兴趣的任务是在存在测量噪声的情况下识别突变点的位置。这些变化点可能出现在一个测量通道中，也可能以相关的方式出现在多个通道中。我们开发了一种名为 MULLR（多通道对数似然比测试）的新分析管道，用于跨多个测量通道执行联合变化点识别。作为最早适用于多通道单分子时间追踪的算法之一，MULLR 经过专门设计，无需模型，可以处理具有不同测量统计数据的通道。我们在模拟和实验数据上演示了 MULLR，并将 MULLR 与替代数据处理策略进行了比较。这项工作进一步增强了 ABEL-FRET 在单分子水平上感知生物分子过程的能力。 b) Cas9 全酶组装过程中 gRNA 构象的单分子解剖。生物分子通过一系列功能状态的循环来发挥其功能。为了更好地理解结构-功能关系，在功能状态的连续阶段探测结构非常有意义。我们最近使用 ABEL-FRET 平台在单分子水平上探测 CRISPR RNA (crRNA) 组装成 Cas9 全酶时的 3 端结构。对于每个分子，其组装状态均通过流体动力学分析明确确定，并通过一对策略性放置的 FRET 染料探测其 3 端结构。引人注目的是，我们发现了组装途径的每个阶段的结构异质性和动态，即crRNA、指导RNA（gRNA或crRNA-tracrRNA杂交体）、Cas9-gRNA复合物和Cas9-gRNA与底物DNA结合，凸显了RNA结构多样性的重要性。目前的工作重点是使用 RNA 结构预测工具生成与单分子 FRET 测量一致的结构模型，并设计 Cas9 全酶组装的合理途径。这项工作有可能揭示 Cas9-gRNA 识别的基本生物物理原理