leotide potency is reduced by point mutation in its target site. Schematic of the native and mutant siRNA 3570 target sites PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713189 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 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 4676 or LDM4676. Transfecting cells with different amounts of LDM4676inv resulted in increased HCV replication regardless of the presence or absence of the mutation in the siRNA 3570 target site. In contrast to LDM4676, the inhibitory activity of LDM3570 was clearly reduced by the mutation. At concentrations up to 62.5 nM, the control oligonucleotide LDM3570inv was unable to suppress HCV replication, and its effects were not diminished by the mutation in the siRNA 3570 target site. 221244-14-0 Therefore it can be concluded that at these concentrations the potency of LDM3570 was indeed specifically reduced by mutation of its PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713490 target site. These findings are consistent with the antisense mechanism of action of modified oligonucleotides; furthermore, the data indicate that the observed suppression of HCV replication was not caused by side effects of the ASOs. However, at concentrations 125 or 250 nM, LDM3570inv exhibited toxicity and inhibited HCV replication. Incorporation of 8-oxo-dG residues into LNA/DNA gapmer oligonucleotides has no negative impact on their antisense potency At the highest concentrations, LDM4676 and LDM3570 visibly damaged the transfected cells. Although these ASOs lack the nucleotide sequence descriptors characteristic of hepatotoxic LNA/DNA gapmer oligonucleotides, cytotoxicity may result from additional factors and their combinations. These factors include the transfection protocol, the presence of modified nucleobases and the cytotoxicity of the transfection reagent. As cytotoxicity represents an obstacle for more detailed studies, more suitable transfection methods were sought. Huh7 cells and their derivatives were difficult to transfect with DNA or LNA/DNA gapmer oligonucleotides without causing cell damage. Therefore, the results obtained using different amounts of six transfection reagents and reverse- or directtransfection protocols were compared. Based on the results of this comparison, an optimized Lipofectamine 2000-mediated reverse-transfection protocol was selected that enabled the delivery of siRNA and ASOs into 7075% of the cells. Further increases in transfection efficiency required higher amounts of transfection reagent, which resulted in decreased viability of transfected 
cells. Thus, in subsequent experiments, the reduction of HCV replication to 25 30% of its original level roughly corresponded to complete suppression of HCV replication in every siRNA- or ASO-transfected cell. Even using the selected protocol, higher concentrations of LD467