Abstract/Summary

DNA triplexes, formed by the binding of a triplex-forming oligonucleotide (TFO) within the major groove of a duplex, have been shown to have potential in gene editing and DNA nanotechnology applications. Recently, metal complexes, including ruthenium polypyridyl intercalators, have been widely explored for their distinctive DNA recognition properties and ability to induce site-specific DNA cleavage. Structural information, showing how ruthenium complexes can interact with DNA triplexes, is required to aid the development of compounds capable of selectively targeting and stabilising triple helical structures. This thesis reports solution and crystal-phase characterisation of the binding of ruthenium polypyridyl complexes to DNA triplexes, including the first crystal structure of a complete triplex with intercalated Ru-dppz complexes. UV thermal denaturation experiments were used to assess triplex stability under various conditions related to those used for crystallisation. This included pH (4.0 to 8.0), different cations (Na+ , Mg2+, Ca2+, Sr2+) and spermine, all of which are known to influence triplex thermodynamic stability. The presence of Mg2+ increased the Tm of intermolecular triplexes by ~5 °C and intramolecular triplexes by approximately 10 °C, compared to in the absence of magnesium ions. The observed stability profiles provided valuable guidance for the selection of systems to take forward for crystallisation and structural analysis. The stability and binding preferences of both enantiomers of [Ru(phen)2(dppz)]2+ were then explored in solution by systematically extending the duplex component of a model triplex system. Spectroscopic analysis, including fluorescence spectroscopy and circular dichroism, revealed the -enantiomers bind to terminal CG and TA steps of the extended duplex. While the -enantiomer exhibited fluorescence emission consistent though all the extended systems, stabilisation of the triplex (with a Tm of +1.2 °C) was only observed with CG extensions, suggesting intercalation by the complex adjacent to the terminus of the TFO. Crystallisation of a unimolecular TFO led to the first high-resolution (2Å) X-ray crystal structure of a complete DNA triplex with intercalated ruthenium polypyridyl complexes. Two - [Ru(phen)2(dppz)]2+ complexes intercalated into the minor groove of the DNA triplex, adjacent to T-A:T triplets, separated by a Watson-Crick base pair. This violates the neighbour exclusion prin- ciple due to binding in adjacent DNA steps. Two -[Ru(phen)2(dppz)]2+ complexes also intercalated into TA/TA steps within a DNA duplex cross-over region between symmetry-related triplexes. Crystallisation screening, using the sequences studied in chapter 2, yielded additional crystal structures. A second structure, determined to near-atomic resolution (1.2 Å) revealed for the first time how Ru-dppz complexes can intercalate into the major groove of the underlying duplex, excluding the TFO from the crystal lattice. The intercalation of -[Ru(TAP)2(11-CN-dppz)]2+ in the TA/TA steps into the duplex major groove provides insight into the stacking requirements, as well as the dppz-moieties required to achieve major groove intercalation. Finally, a crystal structure resulting from the self-assembly of a G-rich TFO was obtained. This demonstrated that the TFO could assemble into a G-quadruplex in the presence of K+ . The crystal structure, determined to 1.15 Å resolution, featured G-tetrads, T-tetrads and a novel T:G octaplet motif, at the interface between two non-symmetry equivalent quadruplexes. Overall, these findings provide insights into the intercalation of ruthenium complexes within DNA triplexes, highlighting novel structure formation while emphasizing the importance of careful TFO and DNA triplex design for future studies.