nd sequenced using Sanger sequencing. Transfection of cells with siRNAs or ASOs For RNAi-guided screening, Huh-luc/neo-ET cells were reverse-transfected with siRNAs using Lipofectamine RNAiMAX reagent. Luc activity was measured 48 h post-transfection using reagents and protocols from Promega. The total protein content in the cell lysates was measured by Bradford micro-assay. Lipofectamine 2000, Lipofectamine RNAiMAX, Lipofectamine LTX, DOTAP, FuGENE HD and TurboFect reagents were used to optimize the transfection of Huh-luc/neo-ET cells. Various amounts of these reagents and forward- or reverse-transfection protocols were used to deliver ASOs conjugated to Alexa Fluor 568 into the cells. The transfection efficiencies were 7 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication analyzed using an LSRII flow cytometer. Cytotoxic effects were observed using a Nikon Eclipse confocal microscope. Quantitation of the inhibitory effects of ASOs Cells were collected and lysed at selected time points. The total protein content in the lysate and Luc activity were measured. For the normalization of HCV replication, which is PP 242 proportional to Luc activity, the following calculations were performed. First, to enable the comparison of the average Luc activity per living cell, the total protein content of the cells was used to normalize the Luc activity as previously reported. Thus, the HCV replication signal was expressed as relative light units per microgram of protein. Second, the obtained normalized Luc values were divided by those obtained for the negative controls: “-” siRNA- or mock-transfected PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713490 cells. The averages and standard deviations of seven independent experiments were obtained. Subsequently, the dimensionless average values were fitted with a four-parameter dose-response equation using Prism 5 to estimate the effective concentration 50 values. Results Thermal stability of the all-DNA ASO:RNA duplex is reduced upon incorporation of 8-oxo-dG residues 8-oxo-dG residues have been reported to destabilize ASO:DNA duplexes. However, ASOs are generally used to target RNA rather than DNA molecules. To 
analyze the effects of 8-oxo-dG residues on the binding of ASOs to DNA and RNA molecules, a set of all-DNA oligonucleotides was prepared in which none, one, or two of the centrally located dG residues were substituted with 8-oxo-dG residues. For comparison, a set of ASOs containing 5-OH-dC residues was prepared because of the similarity to 8-oxo-dG; the 5-OH-dC minor tautomeric form was predicted to have abnormally strong bonding to dG residues. Nevertheless, the introduction of 5-OH-dC residues simultaneously into ASO and target DNA also results in decreased ASO:DNA duplex PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710694 stability. All the oligonucleotides were 21 nt long and had identical sequences. Duplex formation between these oligonucleotides and their NH2 = 5′ amino modifier C6; + = prefix for LNA; X = 5-OH-dC; Y = 8-oxo-dG. doi:10.1371/journal.pone.0128686.t002 8 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication targets was monitored by FRET. In this setup, any difference in the Tm of the formed duplexes is attributable to the presence of the 8-oxo-dG or 5-OH-dC modifications. When the 8-oxo-dG modification was introduced, the Tm values of both the ASO:DNA and ASO:RNA duplexes were reduced by ~1.61.8C compared to those of the duplexes formed by control oligonucleotides. Increasing the number of 8-oxo-dG modifications resulted in further reduction of the Tm by ~1.6C for ASO:DNA and by ~2.
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