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

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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.