Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus

Abstract

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 K d of 990 pM in contrast to original pool RNAs with a K d of >1 µM. 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.

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

“…These were similar to those employed previously to investigate aptamer affinity (Bae et al 2002;Hwang et al 2004). 3Dpol protein was biotinylated and immobilized onto streptavidin-coated magnetic beads (as above).…”

“…Binding affinities of the RNA aptamers for the 3Dpol protein were measured using a bead-based immobilization/ precipitation assay that we have developed (Robinson et al 2006), similar to a method described previously (Bae et al 2002;Hwang et al 2004). Increasing amounts of biotinylated 3Dpol were immobilized onto streptavidin beads before incubation with a-32 P UTP radiolabeled RNA aptamers.…”

RNA helicases in infection and disease

“…Screening of chemical libraries has identified limited numbers of weak helicase inhibitors. 212 HCV helicase activity is inhibited in vitro by recombinant antibodies, 213,214 by RNA aptamers 215,216 and by peptides. 217,218 Targeting the nucleotide binding site of HCV-NS3 has proven exceedingly difficult, 219 although nucleotide-mimicking competitive inhibitors have been identified.…”

Understanding Helicases as a Means of Virus Control

“…An aptamer that bind HCV helicase have also been directly selected using the helicase domain of the NS3 protein as the bait in the SELEX procedure [195]. This aptamer (called SE RNA) folds to form four stem loops with GC pairs that are similar to the stem loop located at the 3'-terminal of the negative strand HCV RNA.…”