Beclin-1 expression level was determined by Western blot analysis

dent experiments. The effects of the oligonucleotides on HCV replication are shown on the y-axis. Transfection and normalization of Luc activity were performed as described for Fig 2B. The obtained values were subsequently normalized to those from mock-transfected control cells, which were set to 100%. The values for siRNA 4676, LD4676, and LDM4676 were fitted with a four-parameter dose-response equation; estimated EC50 values are shown in The obtained data revealed that D4676 did not considerably reduce HCV RNA replication, confirming a previous observation that the all-DNA ASOs are not efficient inhibitors. siRNA 4676 had an EC50 of 0.13 nM, whereas the EC50 values for LD4676 and LDM4676 were approximately 50-fold higher. Interestingly, despite the roughly equal mean EC50 values of CI = 95% confidence interval; R2 = goodness of a four-parametric nonlinear regression curve fit; ND = not determined. doi:10.1371/journal.pone.0128686.t004 13 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication LD4676 and LDM4676, the shape of the LD4676 inhibitory curve at higher concentrations was linear rather than non-linear, which was also evident by the goodness of fit with the variable slope non-linear regression curve. Consequently, at the highest concentrations, LDM4676 inhibited the HCV replication signal to a approximately 27% residual level. As approximately 25% inhibition was achieved with siRNA 4676, which is capable of nearly completely inhibiting HCV replication in positively transfected cells, it was calculated that 100 nM LDM4676 suppressed HCV replication by at least 95% in ASO-transfected cells. In contrast, the residual HCV replication level in the presence of the highest concentration of LD4676 was 42%, indicating that HCV replication in ASO-transfected cells was inhibited by no more than 80%. To confirm that the observed inhibitory effects were sequence-specific, control oligonucleotides with inverted sequences were transfected into Huh-luc/neo-ET cells. Importantly, despite an observation of mild cytotoxicity at the highest concentrations, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19696752 none of the control oligonucleotides inhibited HCV replication; these data indicated that the observed effects of the ASOs targeting the siRNA 4676 site were sequence-specific. These results also demonstrated that under certain conditions, LDM4676 might be a more efficient inhibitor of HCV RNA replication than LD4676. However, in general, the incorporation of 8-oxo-dG residues did not result in significant gains in antisense potency in a cell-based HCV replication assay. 8-oxo-dG residues reduce the Tm of the LNA/DNA gapmer ASO:RNA duplex but have little effect on duplex formation The Tm values of the LDM4676:DNA and LDM4676:RNA duplexes were determined and compared to the Tm values of duplexes composed of non-modified all-DNA ASO or LNA/DNA gapmer ASO . Numerous studies have shown that the incorporation of LNA residues strongly increases the binding of ASOs to their targets. Consistent with this, the melting temperature of the LD4676:DNA duplex was 20C higher than the Tm of the D4676:DNA duplex. The effect of LNA residues on the Tm of the ASO:RNA duplex was even more prominent: the Tm increase was A-83-01 price greater than 30C at all analyzed target RNA concentrations. Consistent with the results obtained for all-DNA ASOs, the incorporation of 8-oxo-dG residues reduced the Tm PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698726 of duplexes of LNA:DNA gapmer ASOs with both DNA and RNA targets. For both targets, the decrease in Tm was between 5 and 10C. To inve