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Title: Development of metal complexes and affinity tags that interact selectively with G-quadruplex nucleic acids
Author: Stafford, Verity
ISNI:       0000 0004 5994 1753
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
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Guanine-rich nucleic acids can fold into non-canonical DNA secondary structures called G-quadruplexes. Bio-informatic studies have established that there are ca. 370,000 guanine-rich sequences in the human genome that can potentially form quadruplex DNA. Interestingly these sequences are not randomly distributed; they are present in the telomeres as well as the promoter region of certain oncogenes (such as c-Myc). Due to their biological relevance, G-quadruplex sequences have emerged as potential targets for the development of novel anticancer drugs. Recent interest in obtaining direct, physical evidence for the presence of G-quadruplex structures has fuelled research in the development of synthetic molecules and biomolecules for sensing quadruplex structures within a cellular environment. In this thesis a series of bi-metallic complexes (with platinum, copper and zinc) have been prepared and evaluated as G-quadruplex DNA (Htelo and c-Myc DNA) binders. The metal complexes were designed to possess a metal(II)-terpyridine core capable of π-π stacking with the G-quartet of quadruplex DNA and a metal(II)-cyclen based side arm to provide additional interactions with the loops, grooves and phosphate backbone of DNA. Both homo- and hetero-metallic complexes were prepared. By chelating different metals to the terpyridine and cyclen modalities, different parts of the quadruplex scaffold could be favourably targeted. Once prepared, the ability of the complexes to π-π stack in solid and solution state was probed using X-ray crystallography and 1H NMR spectroscopy. This gave an insight into their propensity to π-π stack with quadruplex DNA. Complex-DNA (quadruplex and duplex) interactions were then studied using a number of biophysical techniques. Fluorescence intercalator displacement (FID) assays, circular dichroism (CD), fluorescence resonance energy transfer (FRET) and UV-vis spectroscopic studies were used. Overall, the studies showed that the bi-metallic complexes displayed high affinity and selectivity towards quadruplex DNA. The performance of the metal complexes in cells was also investigated. The MTS assay was used to determine the toxicity of the complexes towards two cancer (U2OS and SHSY5Y) and one normal (GM0575) cell line. The IC50 values showed the potential of the metal complexes as anti-cancer drugs. Furthermore, immunoblotting was used to probe the ability of these metal complexes to regulate c-Myc oncogene expression. Finally, resin bound G-quadruplex binders (based on platinum-terpyridine and nickel-, vanadium-, zinc-, and copper-salphen complexes) were designed and synthesised. The ability of the resins to bind and isolate (pull-down) fluorescently labelled quadruplex nucleic acid sequences from solution (in the absence and presence of competing biomolecules) was investigated. The affinity tag based on the cyclic amine functionalised nickel(II)-salphen complex gave the most promising results. This affinity tag was able to bind and isolate quadruplex forming DNA and RNA sequence in the presence of excessive amounts of competing biomolecules (including duplex DNA, tRNA, and BSA protein). Furthermore the affinity tag was able to release the bound DNA and RNA sequences upon incubation with denaturing reagents. Therefore the affinity tags presented in this thesis could be employed, after further optimisation, to identify novel quadruplex forming DNA and RNA sequences.
Supervisor: Vilar, Ramon ; Mann, David Sponsor: Engineering and Physical Sciences Research Coucil ; Institute of Chemical Biology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral