Nucleic Acids as Drug Targets -Interactions of Platinum Complexes, Polyamines, and siRNA with DNA and RNA Models

Drugs targeting nucleic acids were studied in DNA and RNA model systems. The effects were investigated with respect to structural and thermodynamic influence, kinetics, and biological function. The drugs employed in this thesis were anticancer active platinum(II) complexes, polyamine derivatives, and siRNA. Structural changes were determined by use of chemical probing and circular dichroism (CD), thermodynamic properties were determined by use of UV-vis spectroscopy, kinetics determined by use of HPLC and chemical probing, and biological activity was measured with respect to suppression of protein production in Rabbit Reticulocyte Lysate (RRL) and in in vitro cell cultures. Formation of covalent adducts between Pt(II) and DNA are believed to be responsible for the anticancer activity of cisplatin. In the present work, the kinetics for the adduct formation reaction was studied as a function of salt concentration with three different platinum complexes as metallation reagents. It was found that with hairpin structures as targets, DNA and RNA are metallated with similar rates. Typically, the platinum(II) complexes formed covalent adducts with the most accessible guanine located in the hairpin loop region. Platination caused the DNA hairpin to change conformation from the natural B-DNA and adopt a Z-like conformation. Further, platination of both DNA- and RNA oligonucleotides were found to destabilize hairpin and duplex structures. Typically, platination of the middle position of a 13-21 nucleotide long duplex resulted in ca 10 °C decrease of the melting temperature (tm) while platination at the end position only decreased tm by 2-3 °C. Addition of naturally occurring polyamines gave rise to an increase of the tm by ca 10 °C. In contrast, polyamines with substituents on the central nitrogen decreased tm significantly. Finally, the efficacy of four different siRNAs targeting the AU-rich region in the 3' UTR of Wnt-5a, was studied. Significant down regulation of protein production was obtained with addition of siRNA in the ?M- and nM range, for RRL and cell cultures respectively. Similar trends were observed with respect to protein suppression levels when monitored in RRL and in cell culture systems. Platination of the siRNA sense strands was found to be compatible with significant activity and combined with reduction of off-target effects