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Subject: search.filters.subject.Quadruplex nucleic acids

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    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
    (Massey University, 2023) Chilton, Bruce
    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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    Targeting DNA secondary structures using chemically modified oligonucleotides : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry, Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Su, Yongdong
    Chemical modifications bring in additional features to oligonucleotides (ONs), including enhanced stability against nucleases, increased binding affinity towards DNA or RNA, improved cellular uptake, etc. This Thesis describes several strategies and chemical modifications used for targeting DNA duplexes and G-quadruplexes. We introduced a pyrene analogue, (R)-1-O-[2-(1-pyrenylethynyl)phenylmethyl]-glycerol, called ortho-TINA (twisted intercalating nucleic acid) monomer into a native duplex DNA. The affinity of ortho-TINA modified strands was low to each other, whereas the affinity of ortho-TINA sequence towards complementary DNA was increased. This property of ortho-TINA duplex was applied for targeting native duplexes in a sequence-specific manner using a process called dual duplex invasion (DDI). The speed of DDI is increased with the increased number of ortho-TINA pairs present in the duplex, as well as with the rise of temperature from 4 to 37 ℃. However, DDI against duplexes longer than the probe is compromised. To improve the kinetics of DDI, we designed and synthesised DNA probes with zwitterionic moieties, 4‐(trimethylammonium)butylsulfonyl phosphoramidate groups (N+), in which the negatively charged phosphate is neutralised by the positively charged quaternary amine. We assume that several N+ moieties in the DNA probe should reduce the electrostatic repulsion between the probe and the target duplex, and in this way, enhance DDI. However, no improvement of kinetics was achieved using N+ modifications in the probe alone and in combination with ortho-TINA monomers. Application of ONs bearing N+ modifications was explored further in parallel DNA triplexes and G-quadruplex. The initial stage of assembly of N+TG₄T proceeded faster in the presence of Na⁺ than K⁺ ions, which contrasted the trend observed for unmodified sequences, and this process was independent of the ionic strength in solution. We also evaluated several other phosphate modifications alongside for a comparison with our N+ modified DNA. Finally, several directions of future work are proposed based on the results obtained in the present Thesis.
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    Characterising a biologically relevant protein-G4 interaction : HP1α and TERRA : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Roach, Ruby
    Our genetic material is intricately folded and protected through the formation of a compact nucleoprotein complex, termed heterochromatin. In addition to controlling the expression of genes, heterochromatin formation is important for the structural integrity of our genome, specifically for the centromeres, the central attachment point of our chromosomes, and also the telomeres, the ends of our chromosomes. The way in which the heterochromatin in these areas is formed and maintained is though the recruitment and binding of the pivotal chromatin regulator, Heterochromatin Protein 1α (HP1α). The current model that explains how and why HP1α is recruited to, and maintained at, regions of constitutive heterochromatin is simple: HP1α binds post-translational modifications on histones (eg. H3K9me3). However, this binding is not high affinity, therefore may not be the sole determinant in HP1α localisation. At the centromeres, it has been shown that a long non-coding RNA transcribed from the peri-centromeres is responsible for recruiting HP1α to this crucial region. At the telomeres, it is proposed that the long non-coding RNA transcribed from the telomeric DNA is responsible for this same purpose. Because of its guanine-rich sequence, it is able to form a non-canonical nucleic acid structure, the G-quadruplex, which may provide the specificity for heterochromatin formation at telomeres. This TElomeric Repeat-containing RNA (TERRA) has been implicated in telomeric elongation pathways, relating it to the immortalisation of cancer cells. It was found that HP1α can specifically recognise the parallel topology of TERRA, binding with high affinity through HP1α’s unstructured hinge. HP1α was also shown to bind other G-quadruplexes of parallel topology, specifically DNA present in promoter and regulatory regions of many proto-oncogenes, implicating HP1α in potential G-quadruplex-dependent gene expression regulatory mechanisms. The interaction shown here between HP1α and TERRA shows a novel mechanism of telomeric heterochromatin formation, providing crucial insights into telomere maintenance and health in human cells, and furthering our understanding of the role of G-quadruplexes in the epigenome.
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    Development of cross-linking strategies for DNA G-quadruplexes : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Chilton, Bruce
    Chemically modified DNA structures are an important development in the study of DNA properties. They allow the manipulation of biophysical properties of DNA complexes, which can give unique perspectives on the behaviours of DNA and molecules, such as enzymes, which interact with DNA. The use of chemical modification to study G-quadruplex structures has been limited, but has shown potential as a method of controlling their topology and behaviour. We plan to achieve this by functionalising positions within quadruplex forming sequences and using this to create linkages between strands. We predict this will improve the stability of the secondary structure and improve kon, the association rate, of the complex. We discuss several general approaches to G-quadruplex modification, and further discuss specific strategies for carrying out these modifications. One general method for modifying DNA is directly modifying the nucleotides that make up the sequence. G-quadruplexes are primarily composed of guanosine, modification of which is primarily possible at two positions, 2 and 8, due to the hydrogen bond arrangement within the structure. We target the 2- and 8- position of modified guanosine molecules with amine substitutions and Sonogashira coupling processes, respectively. We report the synthesis of sequences containing thiol functionalised nucleotides, and an initial assessment of their biophysical properties. The second general method for modifying DNA is incorporating unique, non-native nucleotides with specific functionalities. This has previously been accomplished by incorporating ligands directly into sequences, which were used to form G-quadruplexes with transition metal ions such as copper(II), nickel(II) and cobalt(II). We aim to synthesise sequences containing a modified sugar and incorporate pyridine instead of a nucleobase. We hope this will provide quadruplexes which more closely mimic the structure of native complexes, improving the previously observed properties of modified G-quadruplexes formed in the presence of transition metal ions. We present the synthesis and biophysical properties of a unique quadruplex forming sequence containing a 4-pyridine ligand and pyrrolidine sugar. The thermal stability and kinetic properties of these modified structures are examined using circular dichroism spectroscopy, demonstrating the change in properties relative to native G4 DNA.