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Targeting Translation in Human Thymidylate Synthase and Hepatitis C


The goal of this study was to investigate selective translational inhibition of the human thymidylate synthase (TS), and the hepatitis C virus (HCV) internal ribosome entry site (IRES). This was done through the targeting of critical RNA structures within the TS messenger RNA (mRNA) and the HCV IRES, by small molecules or peptides. The TS protein-TS mRNA interaction was also explored through the use of chemical crosslinking, and mass spectrometry. The validity of targeting the TS mRNA was examined through the exploration of various TS mRNA stabilized mutants in the context of an in vitro transcription translation assay. Additionally, select compounds and peptides were tested in biological assays for their ability to ablate TS mRNA regulated translation and HCV IRES driven translation. The mechanism of HCV IRES driven translation and inhibition was also explored through the use of in-vitro transcription-translation assays and IRES mutants. Compounds were chosen for testing based on their potential to form multiple hydrogen bonds within an RNA fold, and thereby biased to target structured RNA. Peptides were chosen based on their similarity to known RNA binding peptides, or based on the primary sequence of the TS protein. Compounds that target the TS mRNA were further examined using a 2-aminopurine fluorescence assay and gel- shift assays. To further investigate the respective targets' structures and interactions, macromolecular x-ray crystallography was employed. The TS mRNA is likely dynamic in solution, and less amenable to crystallography than the HCV IRES. To overcome this obstacle, various TS RNA sequences were synthesized, and set in high-throughput crystal screens. The TS protein was also crystallized in the presence of its RNA under two unique conditions in an attempt to capture the TS-mRNA bound state

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