3 research outputs found
Combining a Ru(II) âBuilding Blockâ and Rapid Screening Approach to Identify DNA Structure-Selective âLight Switchâ Compounds
A chemically
reactive RuÂ(II) âbuilding blockâ, able
to undergo condensation reactions with substituted diamines, was utilized
to create a small library of luminescent âlight switchâ
dipyrido-[3,2-<i>a</i>:2â˛,3â˛-<i>c</i>] phenazine (dppz) complexes. The impact of substituent identity,
position, and the number of substituents on the light switch effect
was investigated. An unbiased, parallel screening approach was used
to evaluate the selectivity of the compounds for a variety of different
biomolecules, including protein, nucleosides, single stranded DNA,
duplex DNA, triplex DNA, and G-quadruplex DNA. Combining these two
approaches allowed for the identification of hit molecules that showed
different selectivities for biologically relevant DNA structures,
particularly triplex and quadruplex DNA
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Structural insights into Ruthenium complex-DNA triplex interactions
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 (2Ă
) 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 Ă
) 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 Ă
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
Design, Synthesis and Physicochemical Analysis of Ruthenium(II) Polypyridyl Complexes for Application in Phototherapy and Nucleic Acid Sensing
Current chemotherapeutics exhibit debilitating side effects as a result of their toxicity to healthy tissues. Reducing these side effects by developing chemotherapeutics with selectivity for cancer cells is an active area of research. Phototherapy is one promising modality for selective treatment, where drug molecules are âturned onâ when irradiated with light, reducing damage to healthy tissues by spatially restricting the areas exposed to irradiation. A second approach to improve selectivity is to exploit the differences in cancerous versus healthy cells, such as increased metabolism and/or upregulation of cell surface receptors. Ruthenium(II) polypyridyl complexes are candidates for phototherapy due to their highly tunable photophysical and photochemical properties. The addition of strain to the metal center is a general approach used to render complexes susceptible to light-induced ligand loss. Upon ejection of a ligand, the Ru(II) center is capable of covalently binding biomolecules within cells to produce a cytotoxic effect. The ligands surrounding the metal center are amenable to chemical modification through the incorporation of pendent functional groups as chemical âhandlesâ, allowing for different directing molecules to be attached.
Nucleic acids are important targets for drug discovery, and the development of selective probes to either visualize or selectively damage nucleic acids within the cell is an ongoing area of research. Specifically, G-rich regions are abundant in the human genome, and the presence of G-quadruplexes in telomeres and promoter regions of oncogenes make them potential therapeutic targets. Ru(II) complexes are known to bind nucleic acids, and some have been shown to induce and/or stabilize G-quadruplex Structures. Multiple series of Ru(II) compounds have been synthesized and tested to improve the functional range for Ru(II) complexes for in vivo applications, where they act as âlight switchesâ for DNA. These molecules are âoffâ when in an aqueous environment but turned âonâ in the presence of DNA. Several hit compounds were identified that showed selectivity for specific G-quadruplex structures