Interfering with hepatitis C virus IRES activity using RNA molecules identified by a novel in vitro selection method

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 isolation of efficient anti-HCV internal ribosome entry site (IRES) RNA molecules identified by a new in vitro selection method. The newly developed procedure consists of two sequential steps that use distinct criteria for selection: 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%.

Modular evolution and increase of functional complexity in replicating RNA molecules

“…Computer simulations of structure-based modular evolution could complement in vitro evolution experiments in other ways as well. They can be useful in predicting or analyzing the results observed in experiments in which an RNA module is added to a preformed ribozyme or aptamer (Kumar and Joyce 2003;Romero-Ló pez et al 2005), and in optimizing the effect of the combination of modules to develop allosteric ribozymes with relevant biotechnological applications (Komatsu et al 2002;Penchovsky and Breaker 2005). Conversely, the structural invariability of the catalytic domain, which is susceptible to being computationally estimated, could be used as a key criterion in the design of experiments aimed at isolating the core elements of functional RNAs by trimming a longer, in vitro evolved aptamer or ribozyme (Lozupone et al 2003;Wang and Unrau 2005).…”

“…Also, functional motifs have been successfully combined to generate allosteric ribozymes (Tang and Breaker 1997;Komatsu et al 2002), effector-activated ribozymes (Robertson and Ellington 2000), and complex molecules endowed with two activities, such as RNA cleavage and ligation (Landweber and Pokrovskaya 1999;Kumar and Joyce 2003). More recently, a two-step in vitro evolution method was developed that allowed the sequential selection for specific ligand binding and cleavage, so that the evolved catalytic RNAs carried an aptamer domain and a ribozyme one (Romero-Ló pez et al 2005). A different experimental approach was carried out to evolve minimal functional RNAs, leading to the characterization of certain activities performed by individual RNA modules (Lozupone et al 2003;Wang and Unrau 2005).…”

RNA Selection and EvolutionIn Vitro:Powerful Techniques for the Analysis and Identification of new Molecular Tools

“…This allows structural reorganizations that fi nally release the functional catalytic module. In vitro selection (99) procedures designed to identify allosteric ribozymes requires a randomised sequence, which act as sensor, to be attached to the catalytic domain. The effector molecule is mixed with the library under permissive reaction conditions to promote the active folding of the ribozyme.…”

“…Based on this principle, an in vitro selection procedure has been designed for the identifi cation of new inhibitors of the IRES region of the hepatitis C virus (HCV), (Fig. 6), (99). An RNA library was constructed, carrying the hammerhead ribozyme that cleaves the HCV RNA genome at nucleotide 363 (HH363), (67).…”

“…This strategy identifi ed those RNA molecules composed of an aptamer and a catalytic domain which showed effective anti-IRES activity. This innovative approach of combining the features of aptamers and ribozymes in a single molecule would seem to be very promising since greater HCV inhibition is achieved than with the ribozyme or aptamer alone (99). The same strategy might be used to obtain RNA molecules targeting other viral or cellular RNAs.…”