Characterization of the structure and variability of an internal region of hepatitis C virus RNA for M1 RNA guide sequence ribozyme targeting.
Accessibility to folded RNA and low potential of variation in the target RNA are crucial requirements for ribozyme therapy against virus infections. In hepatitis C virus (HCV), the sequence of the 5'UTR is conserved but the highly folded RNA structure severely limits the number of accessible sites. To expand investigation of targeting in the HCV genome, we have considered an internal genomic region whose sequence variation has been widely investigated and which has a particularly conserved RNA structure, which makes it accessible to the human RNase P in vitro. We have first mapped the accessibility of the genomic RNA to complementary DNAs within this internal genomic region. We performed a kinetic and thermodynamic study. Accordingly, we have designed and assayed four RNase P M1 RNA guide sequence ribozymes targeted to the selected sites. Considerations of RNA structural accessibility and sequence variation indicate that several target sites should be defined for simultaneous attack.
Nadal
Anna
A
Servicio de Medicina Interna-Hepatología, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.
Robertson
Hugh D
HD
Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021, USA.
Guardia
Jaime
J
Servicio de Medicina Interna-Hepatología, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.
Gómez
Jordi
J
Servicio de Medicina Interna-Hepatología, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain.
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D043262:Ribonuclease P; D013816:Thermodynamics; D058727:RNA, Small Untranslated
D004279:DNA, Viral; D016337:RNA, Catalytic; D012367:RNA, Viral; D004722:Endoribonucleases; C468368:RPP14 protein, human; D043262:Ribonuclease P; D058727:RNA, Small Untranslated