Novel secondary structures of DNA; and development of a sensitive methodology for capturing DNA/RNA G-quadruplexes from living Drosophila salivary glands

Work reported in this thesis, from three independent projects, highlights: first, a novel DNA secondary structure fold from a neurodegenerative disease-linked repeat sequence; second, a new approach for assembling and reversing a long and 1- dimensional DNA nanostructure. The third and most substantial project reports the development of and biological results from a highly selective and sensitive approach for in vitro and in vivo tagging of DNA and RNA G-quadruplexes. In the first project, a wholly novel higher-order fold of DNA, named as "iCD-DNA", was discovered and characterized. iCD-DNA was found to be formed uniquely by a hexanucleotide repeat expansion sequence, d(C2G4)n, located at the 5' UTR of the C9orf72 gene, causally linked to multiple neurological disorders such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). It was found that incubating d(C2G4)n under mildly acidic conditions and in the presence of non-quadruplex supporting cations (e.g. Li+, Mg2+) gave rise to a distinctive higher order structure whose most striking feature was an inverted circular dichroism (CD) spectrum, distinguishable from the inverted CD spectra of either a left-handed duplex ("Z-DNA") or a left-handed G-Quadruplex ("Z-GQ"). On the basis of CD spectroscopy, gel mobility and chemical footprinting, structural models were proposed for iCD-DNA. In the second project, a new strategy for creating a long (~200-300 nm) and reversible 1-Dimensional DNA nanostructure/ nanowire (1DDN), named "(TQs)n", was designed and carried out. "(TQs)n" incorporates a hybrid of DNA triple and quadruplex helices. In this design, a novel approach for joining together DNA helices (called guanine-rich "glue junctions") was proposed and demonstrated. In the third project, a highly specific and sensitive methodology for uniquely biotin-tagging DNA/RNA G-quadruplexes (by way of their intrinsic peroxidase activity while complexed with heme) was deeply characterised, first, in vitro, and then applied to tag and pull down G-quadruplex forming RNAs and DNAs from living Drosophila larval salivary glands. Preliminary-sequencing data, so obtained, provided initial insights for the potential occurrence of G-quadruplexes in living cells but needs detailed future investigation.