High-Affinity Aptamers to Subtype 3a Hepatitis C Virus Polymerase Display Genotypic Specificity

Abstract

Research into antiviral agents directed at hepatitis C virus (HCV) proteins is 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 genotype 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach used in this study that 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 RNA-dependent RNA polymerase (RdRp) (nonstructural protein 5B [NS5B]) of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 as the partitioning system. Two aptamers, r10/43 and r10/47, were chosen for further studies on the basis of their abilities to bind the HCV RdRp and inhibit polymerase activity. The affinities (equilibrium dissociation constants) of these aptamers for the HCV subtype 3a polymerase were estimated to be 1.3 +/- 0.3 nM (r10/43) and 23.5 +/- 6.7 nM (r10/47). The inhibition constants of r10/43 and r10/47 were estimated to be 1.4 +/- 2.4 nM and 6.0 +/- 2.3 nM, respectively. Inhibition of HCV 3a polymerase was specific for r10/47, while r10/43 also demonstrated some inhibitory effect on norovirus and phi6 polymerase activity. Neither r10/43 nor r10/47 was able to inhibit the RdRp activity of HCV genotype 1a and 1b polymerases. This study is the first description of an inhibitor specific to the HCV subtype 3a polymerase.