Selection of RNA Aptamers that Bind Specifically to the NS3 Protease of Hepatitis C Virus

The RNA genome of human hepatitis C virus (HCV) is translated into a large precursor polyprotein. The NS3 protease of HCV has a crucial role in the processing of the polyprotein into functional viral proteins. We have used an in vitro genetic‐selection strategy to isolate high‐affinity RNA aptamers that bind to the NS3 protein, especially to its protease domain. Starting from a RNA pool that had a random sequence core of 12–18 nucleotides, aptamers that bind specifically to the NS3 protein were selected after 10 rounds of selection and amplification. A single aptamer, 10G‐1, was found predominantly (71%) in the selected pool. This aptamer could bind to the NS3 protein with a binding constant of 650 nM and inhibit the proteolytic activity in vitro. By phosphate‐modification‐interference analysis we showed that the phosphate residues that are critical for the binding of 10G‐1 to NS3 lie within the selected regions of the aptamer and that binding involves electrostatic contacts with the phosphates of regions G28–U34 and A47–A55. The NS3‐binding region in 10G‐1 can serve as a basis for designing more potential inhibitors of the NS3 protein.