lected immediately after mixing the substrate and enzyme or after incubation for 10 s or 0.5, 1, 5, 10 or 20 min. The reactions were stopped by adding an equal volume of 2x stop buffer. The samples were then heated to 95C for 2 min, cooled rapidly on ice and separated by PAGE in native 15% gels. The gels were dried, exposed to a storage phosphor screen and visualized using a Typhoon Trio scanner. Quantitative analyses were performed using ImageQuant TL Software. Analysis of the stability of ASOs in human serum To analyze the stability of D4676, DM4676, LD4676, and LDM4676 in human serum, these compounds were labeled with 33P as described above for RNA oligonucleotides. Five fmol of each 33P-labeled ASO was incubated in human serum at 37C. Aliquots were collected immediately after preparation of the mixtures or after incubation for 0.25, 0.5, 1, 2, 4 or 6 h. Next, 2x stop solution was added to each aliquot. The full-length oligonucleotides and their degradation products were separated and analyzed as described above. The average PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19709857 half-lives of each type of ASO were obtained from three independent experiments by fitting the obtained values to an exponential decay function. HCV replicon cells Huh-luc/neo-ET cells, which harbor the I389/NS3-3’/LucUbiNeo-ET replicon of HCV genotype 1b , and replicon-free Huh7-cure cells were obtained from ReBlikon GmbH. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with penicillin, streptomycin, 0.5 mg/ml G418, 10% fetal calf serum and 2 mM L-glutamine. A variant replicon that encodes for NS3 with Thr54Ala mutation, was constructed using site-directed mutagenesis and designated I389/NS3-3’/LucUbiNeo-ET-T54A. The corresponding cell line, designated Huh-luc/neo-ET-3570mut, was obtained by electroporation of Huh7-cure cells with the corresponding in vitro-transcribed RNAs and selection of antibioticresistant colonies in the presence of 0.5 mg/ml G418. The preservation of the introduced mutation was verified as follows. Total RNA was extracted from Huh-luc/neo-ET3570mut cell line using an RNeasy Mini Kit. Reverse transcription was carried out 6 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication Fig 2. RNAi-guided oligonucleotide target-site selection in the coding region of HCV RNA. Schematic of the HCV genome and the luc/neo-ET replicon. The numbers above the HCV genomic RNA indicate the positions of the start codons for the non-structural proteins NS3-NS5B. Luc/neo, firefly luciferase/neomycin phosphotransferase cassette; E-I, encephalomyocarditis virus IRES element. Inhibitory effects of thirty-two different siRNAs targeting the NS3-NS5B region of the luc/neo-ET replicon. The siRNAs were transfected into Huh-luc/neo-ET cells at a MedChemExpress Aphrodine concentration of 100 nM. At 48 h p.t., the total protein content and Luc activities in cell lysates were determined. The Luc activities were first normalized to total protein content; next, the obtained values were normalized to the value obtained for control cells transfected with non-targeting negative control siRNA, which was set to 1. The y-axis indicates the fold inhibition of HCV replication achieved using the corresponding siRNAs. The error bars represent the standard deviation of three independent experiments. doi:10.1371/journal.pone.0128686.g002 using a First-Strand cDNA Synthesis kit. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710274 HCV-specific cDNA fragment containing the mutation site was PCR-amplified using of primers flanking the mutated region. Obtained PCR products were purified a