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Decoding the Fabric of Life: Nanopore Sensing of Nucleic Acid Structure from the Primary to the Quaternary https://doi.org/10.17863/CAM.115402 https://doi.org/10.17863/CAM.115402 Meng, Siong Chen Keyser, Ulrich Nucleic acids exhibit a great variety of structures at all levels of organisation. These range from chemical modifications on nucleotides at the primary level through non-canonical tertiary structures like triplexes, and to quaternary structures such as naturally occurring ribosomes and artificially induced DNA/RNA nanostructures. The structure of nucleic acids is also intimately linked to their biological function. In this thesis, I leveraged RNA/DNA nanotechnology and solid-state nanopore sensing to detect RNA structural modifications and to characterise the kinetics of nanostructure formation. Additionally, I aimed to directly identify triplexes solely using nanopore sensing. The group’s RNA Single-nucleotide Characterisation and Analysis Nanolatch (RNA-SCAN) system, a type of RNA/DNA nanotechnology, takes advantage of the effect of RNA modifications on duplex binding. This system was able to detect the m5C1407 modification in E. coli 16S rRNA. I developed a two-state model for this system and used it to extract thermodynamic data from various modifications that were used, such as 5-methylcytosine, 2’-deoxyinosine and 2’-O-methoxyethyl . The data acquired agreed well with values from the nearest neighbour model. Furthermore, I validated the assumption that the RNA-SCAN system is in equilibrium during measurements and attempted to quantitatively track RNA-SCAN dynamics. Lastly, I investigated the current signals in nanopores for suspected triplexes in plasmids with GAA/TTC mirror repeats in various topologies, such as supercoiled, nicked and linearised. I did so by comparing their current traces to ones produced by control plasmids with poly (A/T) repeats or no repeats. I demonstrated that there are two distinct populations in the GAA/TTC plasmids compared to one in the control plasmids, and I sought to show that the second population were indeed triplexes. The work here shows the promise of nanopore sensing to characterise nucleic acid structures at various levels of organisation. University of Cambridge 2024-02-09 Thesis Masters Master of Philosophy (MPhil) https://www.repository.cam.ac.uk/handle/1810/379247 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/34d776e1-fe0d-44de-bed1-0b985944d07b/download e92749df7cee8831570623ab27f1fd55 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a6a15e88-b75f-41c8-996c-8ff7a6a8c3ac/download eb9d2d75cbfff5683a827dd2229be5c0 https://creativecommons.org/licenses/by-nc/4.0/