title: Structural insights into Ruthenium complex-DNA triplex interactions
Creators: name: Abdullrahman, Ahmad
Publisher: name: University of Reading
Year: 2024

description: 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 condi�tions 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 stabil�ity. 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 crys�tallisation and structural analysis. 
The stability and binding preferences of both enantiomers of [Ru(phen)2(dppz)]2+ were then ex�plored 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 interca�lated into TA/TA steps within a DNA duplex cross-over region between symmetry-related tri�plexes.
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, ex�cluding 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.