Biophysical characterisation of DNA triplexes for antigene applications
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(Thesis)
Date
2023
Authors
Klose, Jack
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Advisors
Pukala, Tara
Huang, David
Huang, David
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Thesis
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Abstract
Nucleic acids are fundamental biomacromolecules that act as essential carriers of genetic information and play crucial roles in cellular processes of living organisms. The structure and functionality of duplex deoxyribonucleic acid (DNA) is well characterised in biology, however higher-order DNA structures such as DNA triplexes are relatively poorly understood. DNA triplexes are constituted from the canonical double-stranded DNA and a third strand known as the triplex forming oligonucleotide (TFO). The TFO binds to polypurine stretches within the major groove of the duplex via Hoogsteen hydrogen bonding. It has been indicated that these triple helical assemblies have gene repressive traits, making these structures promising for use in antigene technology for the development of new DNA therapeutics. However, the restriction to polypurine sequences limits the available triplex forming sites in gene targets, as well as their stability. Furthermore, evidence for the ability of triplexes to manipulate gene regulation is primarily based on in vitro experiments, leaving numerous aspects to be explored regarding the cellular functions of these structures. This thesis investigates DNA triplex structures using native mass spectrometry (MS) as well as other solution-phase based techniques. It explores the impact of solution conditions, sequence specificity and base modifications on the formation of these non-canonical structures. The findings contribute insights into the manipulation and potential biotechnological applications, including antigene therapeutics, of higher-order DNA structures. Following a review of the current literature surrounding the formation and modification of DNA triplexes and methods for their analysis, Chapter 3 focuses on the study of model parallel triplex systems using native MS, UV-vis spectroscopy and isothermal titration calorimetry. Different solution conditions including salt concentrations and pH, as well as ionization modes for MS were investigated and shown to have a profound effect on the formation of DNA triplexes and the species detected respectively. The use of ammonium acetate makes these triplex systems compatible with mass spectrometry (MS) experiments, although care needs to be taken when selecting salt concentrations for in vitro studies, as over-stabilisation of triplexes is possible when trying to emulate biological conditions. It is well known that sequence specificity is an important aspect to consider for triplex formation. When probing the specific sequences using both positive and negative ionization modes in MS, this led to observations of different species in the respective ionization modes. Although there is no change in the relative abundance of triplex formation it is worth noting for the analysis of all triplex systems using MS in the future. The formation of i-motifs was also a sequence-dependent observation detected in low pH conditions in the alternating cytosine and thymine triplex system, which is known to have pH dependence. These structures cannot be identified by solution-phase techniques, highlighting the advantages of analysing higher-order DNA structures using MS. Triplex target sites are limited in bacterial genomes due to the high chance of having pyrimidine interruptions occurring in the requisite polypurine sequences, which make it increasingly difficult to form DNA triplexes. Chapter 4 investigates the use of dSpacer (2’-deoxyribonucleoside 3’-O-phosphoramidite) and locked nucleic acid (LNA) base modifications to enable triplex formation in such sequences with a pyrimidine interruption. The LNA modification showed enhanced triplex forming capability compared with dspacer and regular DNA bases in the same position. These modifications were also trialled using biologically relevant triplex target sites from a Pseudomonas aeruginosa genome. Again, the LNA-based TFOs proved to be the most effective in overcoming pyrimidine interruptions, indicating that synthetic TFOs are a viable approach to stabilise triplex structures for potential gene targeting applications in vivo. Chapter 5 examines the use of a TFO and cell-penetrating peptide conjugate to target specific triplex forming sequences both in vitro and in vivo as an antigene antibacterial strategy. Using bioinformatics approaches, biologically relevant triplex target sites were identified from a methicillin-resistant Staphylococcus aureus genome. The dual-specificity RNA methyltransferase (rlmN) gene was chosen as the candidate antigene site due to being of sufficient length without any pyrimidine interruptions and the positioning of the sequence within the promoter region of the gene. The rlmN gene is also responsible for the production of the dual-specificity RNA methyltransferase protein and assists with resistance to antibiotics, making it an attractive target for antigene technology. Here, a synthetic TFO-cell penetrating peptide conjugate was constructed as a means of delivering the TFO into bacterial cells. Successful triplex formation was shown using the synthetic conjugate as the TFO by MS and UV-vis spectroscopy techniques. The rlmN TFO-cell penetrating peptide conjugate showed moderate antimicrobial activity but was inferior to current antibiotics when tested against Staphylococcus aureus, indicating that more research is required to successfully deliver the synthetic TFO into bacterial cells. Finally, MS was used to characterise the binding of anthocyanins to DNA triplexes. The interactions between anthocyanins and triplexes have only been previously observed using solution-phase methods and have shown moderate stabilising effects. The binding of Anthocyanins indicates the potential to use these small molecules to aid the stabilisation of DNA triplexes for therapeutic applications. MS experiments indicated that anthocyanins preferentially bind to triplex DNA structures over duplex and single strands. In some cases, two anthocyanin molecules were able to bind to the triplex, once again demonstrating the ability for MS to provide information that cannot be uncovered by techniques such as UV-Vis spectroscopy. The overarching goal to use DNA triplexes for antigene technology is still in early stages of development. However, furthering the understanding of how sequence specificity, synthetic base modification and small molecules can be manipulated to stabilise DNA triplexes will allow for more biological functions and antigene therapeutics to be revealed.
School/Discipline
School of Physics, Chemistry and Earth Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Physics, Chemistry and Earth Sciences, 2024
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