Biol. Chem., Vol. 386, pp. 183–190, February 2005 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2005.023 2005/197 Article in press - uncorrected proof Interfering with hepatitis C virus IRES activity using RNA molecules identified by a novel in vitro selection method Cristina Romero-Lo ´ pez, Alicia Barroso- delJesus, Elena Puerta-Ferna ´ ndez and Alfredo Berzal-Herranz* Instituto de Parasitologı´a y Biomedicina ‘Lo ´ pez-Neyra’, CSIC, Parque Tecnolo ´ gico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla, E-18100 Granada, Spain * Corresponding author e-mail: aberzalh@ipb.csic.es Abstract Hepatitis C virus (HCV) infection is one of the world’s major health problems, and the identification of efficient HCV inhibitors is a major goal. Here we report the iso- lation of efficient anti-HCV internal ribosome entry site (IRES) RNA molecules identified by a new in vitro selec- tion method. The newly developed procedure consists of two sequential steps that use distinct criteria for selec- tion: selection for binding and selection for cleaving. The selection protocol was applied to a population of more than 10 15 variants of an anti-hepatitis C virus ribozyme covalently linked to an aptamer motif. The ribozyme was directed against positions 357 to 369 of the HCV IRES, and the cleavage substrate was a 691-nucleotide-long RNA fragment that comprises the entire HCV IRES domain. After six selection cycles, seven groups of RNA variants were identified. A representative of each group was tested for its capacity to inhibit IRES activity using in vitro translation assays. All selected RNAs promoted significant inhibition, some by as much as 95%. Keywords: anti-hepatitis C virus internal ribosome entry site (HCV IRES) RNAs; catalytic RNAs; hepatitis C virus internal ribosome entry site (HCV IRES); inhibitor RNAs; in vitro selection; RNA aptamers. Introduction The hepatitis C virus (HCV) is the major cause of trans- fusion-associated non-A, non-B hepatitis worldwide. Great efforts have been made to combat HCV infection, but the results obtained to date have not been satisfac- tory (reviewed in Dev et al., 2004). There is a real need to develop new therapeutic agents. Nucleic acids in gen- eral, and RNA molecules in particular, are firm candi- dates. On the other hand, HCV is an attractive target candidate for RNA-based therapeutic strategies since the viral genome is exclusively present as RNA: a ca. 9600-nucleotide-long plus polarity single-stranded RNA molecule (Choo et al., 1989) that codes for a single poly- protein. The initiation of translation occurs via an internal ribosome entry site (IRES). Most of the essential IRES domain is contained in the 59-untranslated region (59- UTR) and it spans as many as 30 nucleotides within the translatable domain (Reynolds et al., 1995; Wang et al., 2000). It folds into a complex, highly conserved second- ary and tertiary structure essential for its activity (Honda et al., 1996). The HCV genome is highly variable, with the 59-UTR showing the highest rates of conservation (Bukh et al., 1992). Several attempts to develop RNA therapeutic agents (mainly ribozymes) against HCV infection have been reported in the literature in recent years, with the 59-UTR and the coding region of the nucleocapsid the targets that have attracted most attention (Wakita et al., 1999; Puerta-Ferna ´ ndez et al., 2003b and references therein). Ribozymes interact with the substrate RNA molecule (e.g., viral RNA) and catalyze site-specific cleavage, but the efficiency of cleavage depends on the ribozyme bind- ing to its target site, and the compact structure of the 59- UTR region renders many potentially cleavable sites inaccessible. We have recently shown that the stable TAR-HIV-1 stem-loop motif can be used for anchoring hairpin and hammerhead ribozymes through a TAR anti- sense domain covalently linked to their 39-end. This yields a hybrid molecule known as a catalytic antisense RNA. The interaction of TAR-antiTAR domains enhances ribozyme efficiency in in vitro HIV-1 RNA processing (Puerta-Ferna ´ ndez et al., 2003a). The present work examines the possibility of taking advantage of the con- served structural motifs of the HCV 59-UTR to anchor anti-HCV ribozymes. Among other applications, in vitro selection strategies can be used to rapidly isolate nucleic acid motifs that efficiently bind specific ligands (reviewed in Breaker, 1997; Wilson and Szostak, 1999). These strat- egies have been applied with differing success for the identification of high-affinity aptamers that bind to spe- cific domains of the HCV IRES (Aldaz-Carroll et al., 2002; Kikuchi et al., 2003; Tallet-Lopez et al., 2003). The aim of the present work was to identify RNA mol- ecules that specifically inhibit HCV IRES activity. To this end, a new in vitro selection method for the isolation of hybrid catalytic RNAs was developed. This method is composed of two sequential selection steps: selection for binding and secondly selection for cleavage. The RNA molecules identified carried an aptamer domain that encourages efficient binding to the HCV IRES covalently linked to a hammerhead ribozyme. In in vitro translation assays, the selected RNAs efficiently inhibited IRES function. Results and discussion In vitro selection of anti-HCV RNAs An innovative in vitro selection method was designed in an attempt to isolate anti-HCV RNAs interfering with HCV