5C for ASO:RNA duplexes. Thus, destabilization resulting from the insertion of 8-oxo-dG modifications occurs not only for ASO:DNA duplexes but also for ASO:RNA duplexes. In contrast, duplexes formed by oligonucleotides with one or two 5-OH-dC residues had nearly the same Tm as duplexes formed by unmodified control oligonucleotides. Thus, the introduction of the 5-OH-dC modification into an ASO alone had virtually no effect on the stability of duplexes formed with DNA or RNA. RNAi-guided selection reveals potential ASO target sites in the coding region of HCV RNA Known targets for ASOs are located in a 350-nt region at the 5′-terminus of HCV RNA. Clearly, targeting a region that comprises less than four percent of the virus genome is a Amezinium metilsulfate web pubmed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710468 bottleneck that hinders the development of the most efficient ASOs. RNAi technology was used to search for highly accessible target sites in the HCV coding region. Twenty-eight different siRNAs targeting the HCV genome were designed. Each siRNA had a 19-nt duplex region with 2-nt 3′-overhangs. In addition, four siRNAs that were previously reported to efficiently inhibit HCV replication were used for comparison. Nontargeting siRNAs and a combination of siRNAs against a sequence encoding Luc marker were used as negative and positive controls, respectively. The level of Luc activity in Huh-luc/neo-ET cells is directly proportional to the copy number and replication efficiency of the HCV subgenomic replicon, making it an efficient tool for analyzing the anti-HCV efficiencies of the obtained siRNAs. At a concentration of 100 nM, the majority of the designed HCV-specific siRNAs induced less of an effect that the positive controls. Moreover, similarly to the negative control siRNA, several siRNAs did not have any effect on HCV replication. The effects of three siRNAs were comparable to those of the positive controls, and two siRNAs were more potent. As the high inhibitory potential of an siRNA indicates the accessibility of the corresponding target sites, it was concluded that RNAi-guided screening enabled the selection of several potential ASO target sites in the HCV coding region. However, an all-DNA ASO based on the sequence of the guide strand of siRNA 4676 was essentially unable to suppress HCV replication. Therefore, HCV-specific 21-mer LNA/DNA gapmer oligonucleotides that contained five LNA monomers at each end and three modified residues in the DNA region were designed. As the target site of siRNA 4676 contained three C-residues in its central region, it was targeted by ASOs containing three 8-oxo-dG nucleotides. The only PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19711918 mutation in the selected target sites, for which the viability of the mutant replicon has been previously demonstrated, is located in the target site of siRNA 3570 and results in a Thr54!Ala change in NS3. Therefore, this mutation was introduced into the HCV replicon that was used to generate a Huh-luc/neo-ET-3570mut cell line. As the central region of the target site of siRNA 3570 contains only two Cresidues, an ASO similar to LDM4676 could not be designed against this site. Therefore, a control LNA/DNA gapmer containing three 5-OH-dC residues was used instead. Oligonucleotides with inverted sequences were used as controls. Huh-luc/neo-ET and Huh-luc/neo-ET-3570mut cells were transfected with different concentrations of siRNA 3570, siRNA 4676, LDM4676, LDM4676inv, LDM3570 and LDM3570inv. A 10 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication Fig 3. Modified LNA/DNA gapmer oligonuc