Rational Design of Dual-Functional Aptamers That Inhibit the Protease and Helicase Activities of HCV NS3

The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. It is essential for HCV proliferation and is therefore a target for anti-HCV drugs. Previously, we obtained RNA aptamers that inhibit either the protease or helicase activity of NS3. During the present study, these aptamers were used to create advanced dual-functional (ADD) aptamers that were potentially more effective inhibitors of NS3 activity. The structural domain of the helicase aptamer, #5Delta, was conjugated via an oligo(U) tract to the 3'-end of the dual functional aptamer NEO-III-14U or the protease aptamer G9-II. The spacer length was optimized to obtain two ADD aptamers, NEO-35-s41 and G925-s50; both were more effective inhibitors of NS3 protease/helicase activity in vitro, especially the helicase, with a four- to five-fold increase in inhibition compared with #5 and NEO-III-14U. Furthermore, G925-s50 effectively inhibited NS3 protease activity in living cells and HCV replication in vitro. Overall, we have demonstrated rational RNA aptamer design based on features of both aptamer and target molecules, as well as successfully combining aptamer function and increasing NS3 inhibition.