title: RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications.
abstract: The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.
authors.affiliation: Institute of Technology, University of Tartu, Tartu, Estonia; GeneCode, Ltd., Tallinn, Estonia.
authors.forename: Margit
authors.initials: M
authors.lastname: Mutso
authors.affiliation: Institute of Technology, University of Tartu, Tartu, Estonia; GeneCode, Ltd., Tallinn, Estonia.
authors.forename: Andrei
authors.initials: A
authors.lastname: Nikonov
authors.affiliation: GeneCode, Ltd., Tallinn, Estonia.
authors.forename: Arno
authors.initials: A
authors.lastname: Pihlak
authors.affiliation: Institute of Technology, University of Tartu, Tartu, Estonia; GeneCode, Ltd., Tallinn, Estonia.
authors.forename: Eva
authors.initials: E
authors.lastname: Žusinaite
authors.affiliation: Institute of Technology, University of Tartu, Tartu, Estonia; GeneCode, Ltd., Tallinn, Estonia.
authors.forename: Liane
authors.initials: L
authors.lastname: Viru
authors.affiliation: Institute of Technology, University of Tartu, Tartu, Estonia.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Selyutina
authors.affiliation: GeneCode, Ltd., Tallinn, Estonia; Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia.
authors.forename: Tõnu
authors.initials: T
authors.lastname: Reintamm
authors.affiliation: GeneCode, Ltd., Tallinn, Estonia; Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia.
authors.forename: Merike
authors.initials: M
authors.lastname: Kelve
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Mart
authors.initials: M
authors.lastname: Saarma
authors.affiliation: GeneCode, Ltd., Tallinn, Estonia; Institute of Chemistry, University of Tartu, Tartu, Estonia.
authors.forename: Mati
authors.initials: M
authors.lastname: Karelson
authors.affiliation: Institute of Technology, University of Tartu, Tartu, Estonia.
authors.forename: Andres
authors.initials: A
authors.lastname: Merits
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D020029:Base Pairing; D045744:Cell Line, Tumor; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000254:growth & development; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D006801:Humans; D058990:Molecular Targeted Therapy; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D059367:RNA Cleavage; D034622:RNA Interference; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014779:Virus Replication
chemical_list: D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D012367:RNA, Viral; C477371:locked nucleic acid; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine