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

项目总结：单分子实验通过能够询问单个分子的结构、动力学和功能，彻底改变了生物系统的研究。然而，通常使用的荧光技术受到光漂白的影响，这限制了可用光子预算、观测的准确性和可获得的时间跨度。这篇论文旨在通过使用瞬时结合的DNA探针与各种分子靶标来促进标记的持续交换来避免光漂白。这项工作的第一部分涉及开发一种新型的、抗漂白的单分子Förster共振能量转移(SmFRET)版本，它将用于监测RNA聚合酶(RNAP)在其化学反应(基因转录)期间的实时构象变化，提供前所未有的一般方法来观察复杂的构象图景及其在较长一段时间内与分子机制的关系。在第二步，瞬时结合将用于监测单分子基因在体外的表达，首次允许复杂的构象和功能状态联系在一起，并随后结合到机制模型中。最后，瞬时结合还将用于体内连续的mRNA监测，通过高分辨率定位提供的时间和空间信息与自然生物背景相结合，将有助于我们加深对局部和全球细胞内环境如何影响基因表达的理解；这一能力也将有助于检测抗生素耐药细菌的标记。这些研究不仅将加深对基因表达机制的了解，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，特别是生物物理学和软物质，以及合成生物学的领域保持一致。

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.