Selection of RNA Aptamers That Are Specific and High-Affinity Ligands of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Abstract

In order to find small RNA molecules that are specific and high-affinity ligands of nonstructural 5B (NS5B) polymerase, we screened by SELEX (systematic evolution of ligands by exponential amplification) a structurally constrained RNA library with an NS5B⌬C55 enzyme carrying a C-terminal biotinylation sequence. Among the selected clones, two aptamers appeared to be high-affinity ligands of NS5B, with apparent dissociation constants in the low nanomolar range. They share a sequence that can assume a stem-loop structure. By mutation analysis, this structure has been shown to correspond to the RNA motif responsible for the tight interaction with NS5B. The aptamers appeared to be highly specific for the hepatitis C virus (HCV) polymerase since interaction with the GB virus B (GBV-B) NS5B protein cannot be observed. This is consistent with the observation that the activity of the HCV NS5B polymerase is efficiently inhibited by the selected aptamers, while neither GBV-B nor poliovirus 3D polymerases are affected. The mechanism of inhibition of the NS5B activity turned out to be noncompetitive with respect to template RNA, suggesting that aptamers and template RNA do not bind to the same site. As a matter of fact, mutations introduced in a basic exposed surface of the thumb domain severely impaired both the binding of and activity inhibition by the RNA aptamers.Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family, which affects more than 3% of the world population. About 80% of the infected patients develop liver cirrhosis and, in some cases, hepatocarcinoma (13). Besides interferon-based treatments, effective therapies to counteract this important public health problem are still lacking.The HCV positive-strand RNA viral genome contains a single open reading frame flanked by 5Ј-and 3Ј-untranslated regions. The open reading frame encodes a polyprotein of ca. 3,010 amino acids which is processed into at least 10 mature proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by both host signal peptidases and viral proteases (10,31). In analogy with other positive-strand RNA viruses, HCV replication is supposed to proceed through the synthesis of negative-strand RNA, which is in turn used as a template for the production of genomic RNA molecules. A virally encoded RNA-dependent RNA polymerase (RdRp) is considered one of the key enzymes involved in both steps of HCV replication and is, therefore, a primary target for the development of antiviral drugs. The HCV RdRp activity has been localized in the 66-kDa nonstructural 5B (NS5B) protein (2). In vitro, purified NS5B has been shown to be a processive enzyme (39) capable of transcribing the full-length HCV genome (28) essentially via a snap-back mechanism. Recent studies indicate that NS5B does direct de novo replication, requiring neither an exogenous primer nor a snap-back priming event on a variety of RNA templates (22,30,37,43,44). The lack of specificity toward the HCV RNA genome suggests that NS5B corresponds to the elongation factor...

Aptamers That Preferentially Bind Type IVB Pili and Inhibit Human Monocytic-Cell Invasion by Salmonella enterica Serovar Typhi

“…In order to increase the specificity of selected RNA aptamers, we have utilized a combinatorial library approach by pretreating RNA aptamers with GST-Sepharose 4B before each round of SELEX selection, from the third round to the eighth and final round. Although this combinatorial library approach has been successfully used to isolate aptamers blocking ligand-receptor interactions between eukaryotic cells and viruses (2,5,8) or between eukaryotic cells themselves (17,22,29), the technique has not been applied to the selection of ligands blocking an interaction between bacterial and eukaryotic cells. Our studies indicate that the SELEX approach is a useful tool to study the roles of bacterial proteins in pathogenesis and invasion of host cells, as well as to provide insights into the mechanisms of pathogen-host cell interactions.…”

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

“…Intracellular expression of tandem repeats of the RNA aptamers against NS3 protease was demonstrated to inhibit the HCV protease activity in HeLa cells (Nishikawa et al 2003). Moreover, high-affinity RNA aptamers to HCV NS5B RNA-dependent RNA polymerase were recently isolated that could inhibit the enzymatic activity in vitro (Biroccio et al 2002;Vo et al 2003). Although RNA ligands isolated from full-length HCV NS3 moderately inhibited HCV helicase activity , no studies have been described for the isolation of RNA aptamers directly against HCV NS3 helicase domain.…”

Pharmacoproteomics in drug development

“…Aptamers have been successfully generated against many proteins, a recent example being aptamers against the hepatitis C virus RNA polymerase. 66 Automated selection techniques and analytical methods soon should make it possible to select aptamers against total organismal proteomes. 67 …”