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Show Jennifer Andelin Department of Chemistry Peter A. Beal CONTROLLING HEPATITIS C VIRAL PROTEIN SYNTHESIS WITH SMALL INTERCALATING MOLECULES Jennifer Andelin, (Peter A. Beal) Department of Chemistry One of the specific goals of the Beal Lab is to design small organic molecules that selectively target duplex RNA. Presently, we are studying Pep-tide Acridine Conjugates (PACs), because acridine has been shown to intercalate duplex RNA non-selectively (1). The Beal group has shown that when peptide appendages are added, binding affinity and selectivity increase. When these molecules bind duplex RNA, normal structure of the RNA is altered, rendering it incapable of it's normal function. The genomes of many pathogenic viruses consist of duplex RNA. If one could synthesize small molecules that were capable of disrupting the normal function of duplex or viral RNA, then therapeutic agents containing these small molecules could stop the progression of viral infection. My focus for this project has been to identify if and where there is a PAC binding site on the internal ribosomal entry site (IRES) of the hepatitis C virus (HCV). It has previously been shown that the PAC, 9-anilino-acridine-GKK, has impeded the translation of HCV IRES (2). The direct cause of translation inhibition is unknown. There is evidence however, to suggest that this PAC binds the IRES and not the ribosome (2). In lieu of these results, and our knowledge of translation inhibition by PAC intercalation on other RNA aptamers, it is our hypothesis that the PAC is binding somewhere on the HCV IRES. To hone in on the binding site we conducted experiments on HCV using MPE, a known, non-selective intercalator. Due to structure similarity of MPE and our PACs, we can project that if there is a good MPE binding site, then the RNA potentially has a good binding site for our PACs as welt After transcribing the RNA from a linearized DNA plasmid, the RNA was radioactively 3'end-labeled and used in affinity cleavage experiments. The products of these cleavage experiments were sequenced using Polyacrylimide Gel Electrophoresis. From these studies, we conclude that there are indeed several MPE binding sites on the HCV IRES that appear to be located in the base of the Hid helix and loop. The next step will be to show if there are more specific and efficient PAC binding sites. I have made 3 new PAC derivatives. 9-Anilino-acridine-GKKK-EDTA (Figure 1) will be used in future affinity cleavage experiments. 9-Anilino-acridine-GKKK, and 9-Anilino-acridine-GKKK-Biotin will be used in future foot-printing experiments. My goal is to subject the HCV IRES to these PACs in order to determine if there is in fact a PAC binding site on the IRES. Once this is confirmed further experiments will be conducted to conclude the most efficient PAC, and it's specific binding site. Figure3 9-anilino-acridine-GKKK-EDTA, a PAC 1. Wilson, W. D., And Jones, R. L, Adv Pharmacol Chemother 1981,18,177-22 2. Malina, A., Khan, S., Carlson, C.B., Svitkin, Y., Harvey, I., Sonenberg, N., Beal, P.A., Pelletier, J., FEBS left, (in press) |
