Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine prot ease that provides an essential function in maturation of the virus by clea ving the nonstructural regions of the viral polyprotein. The goal of this w ork was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide Delta NS3. RNA aptamers were selec ted in vitro by systematic evolution of ligands by exponential enrichment ( SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of Delta NS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main cl asses (G9-I, II and III). These classes include the conserved sequence GA(A /U)UGGGAC. These aptamers bind to Delta NS3 with a binding constant of abou t 10 nM and inhibit approximately 90% of the protease activity of Delta NS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In ad dition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for Delta NS3 with a K-i approximate to 100 n M in the presence of P41. These results suggest that the pool of selected a ptamers have potential as anti-HCV compounds. Mutational analysis of the G9 -I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.