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 PD-1/PD-L1 inhibitor 2 site 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 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 oligonucleotide PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19696906 potency is reduced by point mutation in its target site. Schematic of the native and mutant siRNA 3570 target sites in the HCV replicon bound to LDM3570. Huh-luc/neo-ET and Huh- 11 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication luc/neo-ET-3570mut cells were transfected with increasing concentrations of LDM3570, LDM3570inv and siRNA 3570 or LDM4676, LDM4676inv and siRNA 4676. The HCV replication values were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698988 calculated as described for Fig 2B. The obtained values were subsequently normalized to those from mock-transfected control cells, which was set to 
100%. Each panel represents data from one of two reproducible independent experiments. doi:10.1371/journal.pone.0128686.g003 point mutation in HCV RNA that changes the classical A:U base pair in the siRNA guide-strand: target-RNA duplex to the G:U wobble base pair resulted in a marked decrease in the inhibitory efficiency of siRNA 3570. As expected, this mutation did not alter the inhibitory efficiency of siRNA 4
