Georgiou, Elena; (2024) DNA nanoarchitectures to examine membrane structure and dynamics. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Biological membranes form the boundaries of cells and are a fundamental component to all life forms. Membrane fusion is a dynamic process which is crucial for the transport of biological components into cells and organelles. Our understanding of fusion remains incomplete due to the lack of available tools to control fusion. DNA nanotechnology has emerged as a potent tool, providing precise control over membrane morphology and interaction, and insights into structural dynamics of bilayer vesicles. In this thesis, DNA nanotools are fabricated to control membrane fusion and understand the interaction of DNA-lipid hybrid nanostructures. The first part of this work introduces a mechanical DNA nanostructure which changes its conformation from an open to a close state to enable fusion initiation events of attached membrane vesicles. The DNA structure’s open conformation holds two vesicles at a well-defined distance while addition of a trigger closes the structure to induce possible vesicle fusion. In mechanical nanostructures, cholesterol-modified DNA anchors the lipid vesicles to the structure. The DNA structure and vesicle fusion are analyzed with ensemble methods and single-molecule localization microscopy (SMLM). SMLM revealed that the vesicles wobble around the defined anchoring site of the DNA nanostructure. This insight highlights the need to better understand how membrane vesicles anchor to DNA nanostructures and thereby improve the design of tools to control membrane fusion with dynamic DNA nanostructures. The second part of this work uses a DNA nanoprobe (DNP) to better understand the interactions between vesicles and DNA nanostructures. Parameters including DNA shape, number and position of cholesterol anchors in the structure, and lipid composition of membrane vesicles are explored. Ensemble methods and SMLM shows that DNP with a single lipid anchor can discriminate between differently sized POPC vesicles also affected by curvature induced lipid packing defects. This may be exploited to discern diagnostically relevant exosomes. Curvature may also induce lipid packing defects. The second part of this work advances DNA nanotechnology to better control interactions with the bilayer membrane and thereby facilitates the design of nanodevices for vesicle-based research, biosensing, and diagnostics.

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