Doctor of Philosophy
School of Chemistry - Faculty of Science
Talib, Jihan, Interactions of metal complexes with DNA, PhD thesis, School of Chemistry, University of Wollongong, 2008. http://ro.uow.edu.au/theses/780
Electrospray ionisation mass spectrometry (ESI-MS), absorption spectrophotometry and circular dichroism spectroscopy were used to investigate the non-covalent binding interactions of the nickel complexes [Ni(phen)2(L)]Cl2, (L = phen, dpq ,dpqc and dppz) with the 16mer oligonucleotide D2, which has the following base sequence: (GCTGCCAAATACCTCC/GGAGGTATTTGGCAGC). In addition, the extent of unwinding of the negatively supercoiled plasmid pUC9 caused by the nickel complexes, and the extent to which they inhibit in vitro synthesis of mRNA, were investigated using gel electrophoresis. The results of these studies showed that DNA binding strengthened as the size of the unique ligand was increased. Comparison of each of the above sets of results with those obtained from identical experiments performed using the analogous ruthenium complexes [Ru(phen)2(L)]2+ (L = phen, dpq, dpqc, dppz) showed that varying the metal ion had a measurable effect on DNA binding affinity, with the nickel complexes generally interacting more weakly with D2 than the corresponding ruthenium complexes. ESI-MS/MS and in-source collision-induced dissociation experiments were performed using the tetrameric quadruplex DNA molecule Q5 (TTGGGGGT)4 and antiparallel dimeric quadruplex Q2 (GGGGTTTTGGGG)2 in order to determine their gas-phase dissociation profiles. It was found that the gas phase stability of the quadruplex DNA was dependent on its charge state, the number of oligonucleotide strands that make up the quadruplex, and the number of consecutive G-tetrads that it contains. ESI-MS and circular dichroism spectroscopy were also used to examine the non-covalent binding interactions of the octahedral nickel and ruthenium complexes stated above, as well as several square planar platinum complexes with Q5. The platinum complexes studied were [Pt(en)(phen)]2+, [Pt(en)(3,4,7,8-Me4phen)]2+, [Pt(en)(4,7-Me2phen)]2+ and [Pt(5,6- Me2phen)(S,S-dach)]2+. The results obtained from these experiments showed that each of the three groups of metal complexes were able to bind to Q5. In contrast to what was found in experiments involving the duplex DNA molecule D2, the presence of the intercalating dppz ligand in the coordination sphere of both the nickel and ruthenium complexes did not greatly increase their binding affinity towards quadruplex DNA. This observation suggests that intercalative binding interactions may not play as important a role in the binding of metal complexes to quadruplex DNA. ESI-MS was used to analyse mixtures containing the organic drug daunomycin, Q5, and either [Ru(phen)2(dppz)]2+ or [Pt(en)(4,7-Me2phen)]2+, in order to obtain information about the qDNA binding modes of these metal complexes. The affinity of the above two metal complexes towards parallel tetrameric quadruplexes with different lengths was also compared using ESI-MS in an attempt to shed light on whether they bind to the ends of the quadruplexes or in grooves along their lengths. The optimal conditions required to obtain ESI mass spectra of the non-covalent adduct formed between the DNA binding domain of mouse transcription factor PU.1, and a short 10mer DNA molecule containing its 5'-GGAA-3' consensus sequence, were determined. ESI-MS was then used to probe the extent of inhibition of formation of this non-covalent complex caused by addition of [Ru(phen)2(dppz)]2+ or [Pt(5,6-Me2phen)(S,S-dach)]2+. Both metal complexes were shown to inhibit binding of the transcription factor to its DNA recognition site, demonstrating the potential of these complexes for transcription therapy.
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