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Item Understanding intercalative modulation of G-rich sequence folding: solution structure of a TINA-conjugated antiparallel DNA triplex.(Oxford University Press, 2024-01-28) Garavís M; Edwards PJB; Serrano-Chacón I; Doluca O; Filichev VV; González CWe present here the high-resolution structure of an antiparallel DNA triplex in which a monomer of para-twisted intercalating nucleic acid (para-TINA: (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol) is covalently inserted as a bulge in the third strand of the triplex. TINA is a potent modulator of the hybridization properties of DNA sequences with extremely useful properties when conjugated in G-rich oligonucleotides. The insertion of para-TINA between two guanines of the triplex imparts a high thermal stabilization (ΔTM = 9ºC) to the structure and enhances the quality of NMR spectra by increasing the chemical shift dispersion of proton signals near the TINA location. The structural determination reveals that TINA intercalates between two consecutive triads, causing only local distortions in the structure. The two aromatic moieties of TINA are nearly coplanar, with the phenyl ring intercalating between the flanking guanine bases in the sequence, and the pyrene moiety situated between the Watson-Crick base pair of the two first strands. The precise position of TINA within the triplex structure reveals key TINA-DNA interactions, which explains the high stabilization observed and will aid in the design of new and more efficient binders to DNA.Item Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A(American Chemical Society, 2022-11-15) Kurup HM; Kvach MV; Harjes S; Barzak FM; Jameson GB; Harjes E; Filichev VVDrug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to cross-link two distant modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by the engineered C-terminal domain of A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3 inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine, a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar inhibitor of engineered A3BCTD and wild-type A3A (Ki = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.Item 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, YongdongChemical 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.Item Twisted intercalating nucleic acids (TINA) in guanosine-rich oligonucleotides : a thesis submitted in the partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry, Massey University, Palmerston North, New Zealand.(Massey University, 2013) Doluca, OsmanThe main role in the structural diversity of DNA molecules belongs to guanosines due to their array of hydrogen bond donors and acceptors, large aromatic surface and ability to adopt syn or anti conformations. These properties lead to the formation of various DNA topologies such as triplexes or G-quadruplexes by guanosine-rich oligonucleotides. For a long time these secondary structures were mainly considered to be a fascinating phenomenon with little practical use; it was subsequently realised that these structures are likely to be formed under physiological conditions and therefore might be involved in many important biological processes, including genome recombination, telomere stability and regulation of gene expression. Thus, there is a growing interest in development and control of these non-traditional nucleic acid structures. Although the secondary structures of nucleic acids can be controlled to a certain extent by the careful design of oligonucleotide sequence this strategy alone is not always sufficient. In this thesis we investigated how to control the assemblies of guanosine-rich oligonucleotides using a novel tool, twisted intercalating nucleic acids (TINAs). The incorporation of pyrene-containing TINA monomers into guanosine-rich oligonucleotides led to the formation of stable triplexes or G-quadruplexes depending on the position of TINA monomers. In the light of our results, we have established a set of rules that helps to create a desired structure of guanosine-rich oligonucleotides using TINA molecules. In the second half of the thesis we focused on expanding the functionality of TINA conjugated oligonucleotides. In terms of fluorescence, we synthesised several fluorescently-silent triplex-forming oligonucleotides (TFOs) equipped with a dye at different positions in the DNA. Fluorescence properties were strongly dependent on the position of the dye. These fluorescently silent TFOs showed up to an 18-fold increase in fluorescent intensity upon triplex formation. These findings lay the foundation for the future design of artificial DNA sequences for expanding the repertoire of DNA secondary structures and function.