Studies towards thermodynamically stable G-quadruplexes embedded in canonical DNA duplexes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand. EMBARGOED to 15 May 2025.
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Date
2023
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Massey University
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Abstract
DNA is the polymer responsible for the storage of genetic information and,
ultimately, all processes that occur within the cell. Our understanding of DNA structure and function has developed considerably, but some areas are still unclear. In particular, a range of non-canonical DNA secondary structures such as G-quadruplexes (G4s), i-motifs and triplexes, have also been shown to form in genomic DNA sequences and these structures also appear to have a role in genome function.
Better understanding of the interactions of non-canonical secondary structures is hindered by their transient nature in the context of larger DNA structures, making it difficult to accurately study them using in vitro analytical techniques (e.g., NMR spectroscopy, X-ray crystallography, etc.). They are typically less thermodynamically stable than canonical DNA duplexes and are formed within the genome only in equilibrium with many secondary structures, typically favouring the canonical duplex. They can often only be formed reliably under specific conditions in vitro (e.g., single-stranded, low pH etc.). This thesis presents several strategies designed to stabilise non-canonical G4 secondary structures, which are of interest because they are often found in the promoter regions of oncogenes.
The most commonly used existing G4 stabilisation technique utilises
small-molecule ligands which specifically bind to and stabilise G4 structures. This thesis includes an investigation into both a widely used G4-binding ligand and several newly developed ligands, but their potential to block binding sites and disrupt G4 topology makes them less suitable for our intended applications.
Hydrophobic modifications can encourage aggregation of DNA strands and
therefore increase stability of secondary structures. Hydrophobic phosphate modifications in G4s proved effective at disrupting duplexes and stabilising G4s but was limited by coupling efficiency of the modification and resulting difficulties with purification. Intentional mismatches in the G4-forming sequence were introduced by inverting sequence direction or incorporating α-anomers of nucleotides. This strategy was able to completely disrupt duplex formation while preserving G4 structures, but modification sites have to be carefully considered to avoid significant changes in G4 topology. Internal cross-links were incorporated into DNA using modified nucleotides designed for copper(I)-catalysed azide-alkyne cycloaddition. These cross-links prevent G4 structures from unfolding, but the location of these cross-links must also be carefully considered to prevent disruption of the native G4 topology and blocking protein binding sites. All three of these methods present potential routes for stabilising G4s within larger DNA structures. Furthermore, all three modifications could potentially be expanded to stabilise other non-canonical structures, such as imotifs or triplexes.
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Embargoed until 15 May 2025
Listed in 2023 Dean's List of Exceptional Theses
Keywords
DNA, Quadruplex nucleic acids, Synthesis, Ligands (Biochemistry), Dean's List of Exceptional Theses