| Popis: |
Over the past decade, small RNAs have emerged as important regulators of eukaryotic messenger RNAs (mRNAs). Short-interfering RNAs (siRNAs) and microRNAs (miRNAs) participate in 'RNA silencing' pathways that regulate transcription, chromatin structure, genome integrity, translation, and mRNA stability. Both types of small RNAs can be functionally equivalent, but they are distinguished by their origin. Herein, novel protein- and RNA-based tools for studying RNA silencing and small RNAs are described. Detection, purification, and quantification of small RNAs is important for gaining an understanding of the roles of RNA silencing pathways in eukaryotic organisms. This thesis describes the development of the tombusviral p19 protein, which is a suppressor of RNA silencing, as a tool to study small RNAs. The siRNA-binding properties of the p19 protein in vitro were investigated and an assay for high-throughput siRNA detection and quantification was developed. Furthermore, a small molecule library was screened to identify small molecule inhibitors of the p19-siRNA interaction. These studies identified small molecules that act as potent inhibitors of the p19-siRNA interaction by alkylation of cysteine residues. Mutagenesis revealed novel postulates regarding the role of cysteine residues within the p19 protein. The specificity of p19-small RNA interactions was also investigated using fluorescence-based techniques in order to determine the affinity of p19 for irregularly-structured small RNAs. Differential binding affinities of p19 to canonical and irregularly structured small RNAs has implications for the use of p19 as a tool for detection, purification and quantification of small RNAs in vitro and in diverse eukaryotic organisms. In addition to developing protein-based tools to study small RNAs, we also wanted to investigate the effects of target site accessibility on the design of highly effective siRNAs. Thus, the importance of target site accessibility in the design of effective siRNAs against highly-structured targets was investigated using the Hepatitis C virus (HCV) RNA genome as a model. Since siRNA knockdown is hampered by target site accessibility, and current computational approaches are not yet able to reliably predict accessibility for large target RNAs, an in vitro screening approach for target site accessibility was developed. Native HCV target RNA microarrays were used to predict the potency of small RNAs directed against the HCV replicon RNA genome. This technique could be useful for the identification of novel, highly potent siRNA target sites within the large, highly-structured HCV RNA genome. Our results suggest that the highly-structured nature of the HCV RNA genome may impede the design of highly-effective siRNA-based inhibitors for this important human pathogen. In addition, the methodology described here could be extended to other systems, including the highly-structured genomes of other human pathogens. |
