Using single-molecule imaging of transient biomolecular interactions to probe conformational dynamics and gene expression mechanisms.

使用瞬时生物分子相互作用的单分子成像来探测构象动力学和基因表达机制。

• 批准号: 2440758
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2440758
• 关键词: Using; single; molecule; imaging; transient

A summary of the project: Single-molecule experiments have revolutionised the study of biological systems by enabling interrogation of the structure, dynamics, and function of individual molecules. However, commonly employed techniques using fluorescence suffer from photo-bleaching, which limits the available photon budget, the accuracy of observations, and the accessible timespans. This thesis intends to circumvent photobleaching by facilitating a constant exchange of labels using transiently binding DNA probes to a variety of molecular targets. The first part of the work involves the development of a novel, bleaching-resistant version of single-molecule Förster resonance energy transfer (smFRET), which will be used to monitor the real-time conformational changes in RNA polymerase (RNAP) during its chemical reaction (gene transcription) offering unprecedented and general ways to look at complex conformational landscapes and their relation to molecular mechanisms over an extended period of time. In a second step, transient binding will be used to monitor single-molecule gene expression in vitro, allowing complex conformational profiles and functional states to be linked for the first time, and subsequently combined into mechanistic models. Finally, transient binding will also be employed for continuous mRNA monitoring in vivo, where the acquired temporal and spatial information provided by high-resolution localisation combined with the natural biological context will help advance our understanding on how the local and global intracellular environment affects gene expression; this capability will also be instrumental in detecting markers of antibiotic resistant bacteria. The insight gained from the studies will not only further the knowledge about gene expression mechanisms but will also provide general tools for the functional and structural analysis of a broad range of biological systems and mechanisms.This work is aligned with areas within the Healthcare Technologies and Physics Sciences themes, and especially the Biophysics and Soft Matter, and Synthetic Biology.

项目摘要：单分子实验通过探究单个分子的结构、动力学和功能，彻底改变了生物系统的研究。然而，使用荧光的常用技术遭受光漂白，这限制了可用的光子预算，观测的准确性和可访问的时间跨度。本论文旨在通过利用DNA探针与多种分子靶点的瞬时结合来促进标记物的恒定交换来规避光漂白。这项工作的第一部分涉及开发一种新型的抗漂白版本的单分子Förster共振能量转移（smFRET），该技术将用于实时监测RNA聚合酶（RNAP）在化学反应过程中的构象变化（基因转录）提供了前所未有的和一般的方式来看待复杂的构象景观及其与分子机制的关系，在一个延长的时间段。在第二步中，瞬时结合将用于监测体外单分子基因表达，允许首次连接复杂的构象特征和功能状态，并随后组合成机械模型。最后，瞬时结合也将用于体内连续mRNA监测，其中通过高分辨率定位结合自然生物背景提供的获得的时间和空间信息将有助于推进我们对局部和全局细胞内环境如何影响基因表达的理解;这种能力也将有助于检测抗生素耐药菌的标志物。从这些研究中获得的见解不仅将进一步了解基因表达机制，而且还将为广泛的生物系统和机制的功能和结构分析提供通用工具。这项工作与医疗保健技术和物理科学主题内的领域保持一致，特别是生物物理学和软物质，以及合成生物学。