Nucleic Acids as Drug Targets - Influence of Platinum Complexes on Duplex Stability and Binding Kinetics

Abstract: Gaining a detailed understanding of how human cells are affected by metal-based drugs is crucial, since these drugs have shown great potential in cancer treatment. Cisplatin is used to treat many different types of cancer, e.g. testicular, ovarian, melanoma, bladder, non-small cell lung cancer, small cell lung cancer, myelomas and lymphomas. The miRNA expression profiles in different human cancers differ from healthy tissue and can be used for classification. This difference makes it important to put every drug that is going to be used in a certain cancer treatment regime, in the context of the expression profile of the specific cancer type. The strong correlation between germline mutations in BRCA1 and cancer development makes it an essential target to study for creating novel treatment regimes. This thesis has a focus on studies of the clinically well-characterized platinum drugs cisplatin and oxaliplatin, and how these affect the stability and melting behaviour of similar sequence DNA and RNA duplexes. The studied RNA duplexes showed a more pronounced sensitivity towards platination compared to the DNA duplexes. A novel method was developed to monitor the binding kinetics of cisplatin towards the mature form of miR-146a and short RNAs containing biologically important sites with altered base complementarity. Spectral changes and binding kinetics were monitored over time at 38 °C and the observed rate constants were linearly dependent on the concentration of mono-aquated cisplatin. Binding of mono-aquated cisplatin to the studied RNA duplexes was shown to be salt dependent, with an observed variation in reactivity. Different mimics of miR-146a, which is known to be up-regulated in invasive cell lines, were constructed to study the influence on BRCA1 down-regulation in vitro. The mimics included several types of chemical modifications and modifications made to the duplex architecture. The degree of complementarity of the guide strand towards the target mRNA was gradually increased to study and evaluate the corresponding change in silencing capacity. The idea behind the construction of a fully complementary mimic, was to study the possibility of obtaining similar silencing levels as with siRNAs. The mimic having full complementarity towards the mRNA target did indeed show higher silencing activity compared to the less complementary mimics.