Hepatitis C virus (HCV)-encoded nonstructural
protein 3 (NS3) possesses
protease,
NTPase, and helicase activities, which are considered essential for viral proliferation. Thus, HCV NS3 is a good putative therapeutic target
protein for the development of anti-HCV agents. In this study, we isolated specific
RNA aptamers to the helicase domain of HCV NS3 from a combinatorial
RNA library with 40-nucleotide random sequences using in vitro selection techniques. The isolated RNAs were observed to very avidly bind the HCV helicase with an apparent Kd of 990 pM in contrast to original pool RNAs with a Kd of >1 microM. These
RNA ligands appear to impede binding of substrate
RNA to the HCV helicase and can act as potent decoys to competitively inhibit helicase activity with high efficiency compared with
poly(U) or
tRNA. The minimal binding domain of the
ligands was determined to evaluate the structural features of the isolated
RNA molecules. Interestingly, part of binding motif of the
RNA aptamers consists of similar secondary structure to the 3'-end of HCV negative-strand
RNA. Moreover, intracellular NS3
protein can be specifically detected in situ with the
RNA aptamers, indicating that the selected RNAs are very specific to the HCV NS3 helicase. Furthermore, the
RNA aptamers partially inhibited
RNA synthesis of HCV
subgenomic replicon in Huh-7
hepatoma cell lines. These results suggest that the
RNA aptamers selected in vitro could be useful not only as therapeutic and diagnostic agents of HCV
infection but also as a powerful tool for the study of HCV helicase mechanism.