Treatments for the hepatitis C virus (HCV) are currently only partially effective. Research into antivirals directed at HCV viral proteins are commonly based and tested on a single genotype, namely genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach for the isolation of antiviral agents. SELEX allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNAdependent RNA polymerase (RdRp) [non-structural protein B (NS5B)] protein of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 and resultant aptamers cloned from rounds 2, 4, 8 and 10. Sequences of aptamers were aligned to elucidate common motifs and a proportion of the aptamers from rounds 8 and 10 (29/48) were screened for binding ability using the Biacore. The five ???best binding??? aptamers were investigated for inhibition of 3a polymerase activity in an in vitro polymerase assay. Two aptamers, r10/43 and r10/47, were chosen for further studies based on their ability to inhibit polymerase activity. The inhibition constants (Ki) of r10/43 and r10/47 were estimated to be 1.4 + 2.4 nM and 6.0 + 2.3 nM respectively. The affinity (Kd) of these aptamers for the 3a polymerase was estimated to be 1.3 + 0.3 nM (r10/43) and 23.5 + 6.7 nM (r10/47). The estimated inhibition and dissociation constants of these two aptamers are among the best for inhibitory aptamers of the HCV enzymes (polymerase and protease). Inhibition of HCV 3a polymerase appeared to be specific for r10/47, whilst r10/43 also had some inhibitory effect on norovirus and ??6 polymerase activity. This study is the first description of an inhibitor to the HCV subtype 3a polymerase that investigates genotypic specificity of targeted antivirals.
| Identifer | oai:union.ndltd.org:ADTP/215658 |
| Date | January 2005 |
| Creators | Jones, Louisa Alice School of Biotechnology And Biomolecular Sciences, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Biotechnology And Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright LOUISA ALICE JONES, http://unsworks.unsw.edu.au/copyright |
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