Interfering with hepatitis C virus IRES activity using RNA molecules identified by a novel in vitro selection method.
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%.
Romero-López
Cristina
C
Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla, E-18100 Granada, Spain.
Barroso-delJesus
Alicia
A
Puerta-Fernández
Elena
E
Berzal-Herranz
Alfredo
A
D020121:5' Untranslated Regions; D001483:Base Sequence; D030561:Databases, Nucleic Acid; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000302:isolation & purification / Q000494:pharmacology; D014176:Protein Biosynthesis; D016337:RNA, Catalytic / Q000302:isolation & purification / Q000378:metabolism*; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D020121:5' Untranslated Regions; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D012367:RNA, Viral