Structural and chemical studies of RNA as anti-cancer target and signaling molecule
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Ribonucleic acids have been found to have a major role in causation and propagation of diseases. Targeting such therapeutically important RNA can be a method of treatment of various diseases. Two such therapeutically important RNA discussed in this dissertation are telomerase and extracellular microRNA. Telomerase is a ribonucleoprotein whose role has been implicated in cancer. Telomerase catalyses telomere extension by using a portion of its RNA as a template. During this process a RNA/DNA heteroduplex is formed. We have attempted to inhibit telomerase by targeting this heteroduplex using nucleic acid binders such as intercalators. In this dissertation, we sought to understand the mechanism of telomerase inhibition by intercalators, using a structural approach. The affinity of binding of ethidium bromide to the RNA/DNA heteroduplex was examined by photocrosslinking ethidium azide to the telomerase RNA and using a primer extension assay. Ethidium azide had a greater affinity for the RNA/DNA heteroduplex compared to the telomerase RNA alone. Designing specific inhibitors using ethidium bromide as the scaffold proved to be challenge due to the off target binding to other nucleic acids. Thus, the next part of the dissertation dealt with identifying weak inhibitors of telomerase and then increasing the affinity for the target enzyme by developing interactions with the protein portion of the enzyme. Naproxen was used as a scaffold to introduce functionalities which would interact with the inner surfaces of the protein portion of the enzyme. Peptide libraries using naproxen as the scaffold having varying lengths and motifs were designed. The synthesized derivatives of naproxen were tested for their ability to inhibit telomerase in order to obtain a molecule which was a better inhibitor of telomerase than naproxen alone. The final portion of the dissertation deals with identifying and targeting extracellular microRNA released from cancerous cells. MicroRNA are small (22-25 nucleotides) RNA duplex which regulate gene expression by cleaving the target mRNA or by blocking protein formation. We hypothesized that microRNA released in the extracellular fluid behave as signaling molecules. Extracellular microRNA were observed in the media of culturing carcinoma cells. Thus identifying and targeting these extracellular species can be another strategy in cancer therapy.
Table of Contents
Introduction and literature review -- Determining the mechanism of telomerase inhibition by ethidium azide -- Designing of peptide libraries with a weak intercalator as the scaffold -- Design of naproxen based libraries with varying lengths and point of attachment of naproxen -- Introduction: micro RNA processing, role in diseases and detection techniques -- Experiment to observe and identify small RNA in the extracellular media of culturing cells -- Design of media to generically capture extracellular micro RNA -- Designing of a disc capable of specifically capturing extracellular micro RNA from the media of culturing cells -- Summary and conclusions