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

Abstract

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.