Alexandrou, Effrosyni; (2025) Revealing the Detail in i-Motif DNA structures. Doctoral thesis (Ph.D), UCL (University College London).

Text Effrosyni-Alexandrou-PhD.pdf - Accepted Version Access restricted to UCL open access staff until 1 September 2028. Download (14MB)

Abstract

The cellular functions of nucleic acids rely not only on their sequence, but also on their structure. Beyond the most widely known double helix, DNA can adopt other classes of secondary structural motifs, including the cytosine-rich four-stranded i-motifs. Such non canonical DNA conformations are formed in vivo and play a key role in gene expression. To date, crystallographic structural information on i-motifs is limited, and there are no crystal structures of intramolecular i-motifs. This PhD thesis aims to reveal more structural detail about i-motif DNA structures. Chapter 1 gives a general background surrounding i-motif DNA. Chapter 2 describes the process of the first crystallographic determination of an intramolecular i-motif structure present at the insulin gene linked polymorphic (ILPR) region at the insulin gene promoter. The structure revealed base interactions critical to the stability of the ILPR i-motif and thus to insulin expression. It revealed pockets for rational-based drug design and it is a valuable model for solving other i-motifs. Insulin binding to the ILPR i-motif was shown and a large-scale crystallisation trial with multiple C-rich ILPR sequences is also presented. Chapter 3 explores a shorter i-motif present at the promoter proximal region of the nosCRZDFYLX gene cluster for nitrous oxide reductase in Paracoccus denitrificans, critical to the release of nitrous oxide from soil bacteria. Insights on the 3-dimenstional structure of the NOS i-motif were revealed and models are proposed. The introduction of ligands, such as ellipticine in the NOS crystallisations is also presented. A range of novel exciting methods for nucleic acid crystallographic phasing and refinement are explored in both Chapters 2 and 3. Chapter 4 investigates the mechanism of action of the experimental anti-cancer drug QN-302 and its analogue CM03 involving an i-motif and its complementary guanine-rich G-quadruplex structure at the S100P gene, revealing the dynamic interplay between these two structures.

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