The fractions were precipitated and washed with acetone. Depending on the downstream application, the oligonucleotide preparations were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19712481 re-precipitated using ethanol/ammonium acetate or ethanol/sodium acetate. The oligonucleotides used in the subsequent analyses contained only trace quantities of impurities. Mass spectrometric analysis of oligonucleotides was performed as described previously using a Bruker Daltonics Autoflex instrument. Briefly, the matrix solution consisted of 7.8 mg/ml 2,4,6-trihydroxyacetophenone and 12 mM diammonium citrate in acetonitrile/water. An aliquot of the concentrated chromatographic fraction was mixed with the same volume of matrix solution. One microliter of the resultant mixture was deposited on the target and dried in the air. The samples were analyzed in negative ion mode with the linear configuration. Melting curve analysis using Frster Resonance MedChemExpress GW 501516 Energy Transfer Melting curve data were obtained using FRET as described in detail by You and coworkers. Briefly, DNA or RNA oligonucleotides labeled with Cy3 or TYE563 at the 3′-end 4 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication were purchased from DNA Technology or SigmaAldrich. Complementary oligonucleotides were labeled in-house with FITC at 
the 5′-end or purchased with a 5′ FAM label from Exiqon A/S. Hybridization was quantified by FRET between the FITC and Cy3 labels or the FAM and TYE563 labels when they were brought in close proximity due to the formation of a duplex between the probe and its target. The target oligonucleotides were used at concentrations of 25, 50, 100, 200 and 400 nM, and the probe was always used at a concentration of 50 nM. The probe-target interactions were measured in a 384-well optical plate in a volume of 20 l in buffer containing 150 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19709857 mM NaCl and 50 mM Tris-HCl using an ABI7900HT Real-Time PCR instrument. The temperature was rapidly increased to 95C, and the complexes were allowed to melt for 10 min. The obtained mixtures were heated to 95C and allowed to slowly cool to 35C. The appropriate volume of 5x non-denaturing loading buffer containing 50% glycerol, 0.1% bromophenol blue and 0.1% xylene cyanol was added to the samples, after which ASO:RNA duplexes were purified and quantified as described previously. To analyze the spontaneous formation of duplexes between target ssRNA and ASOs, 5 fmol of 33P-labeled ssRNA was mixed with 50 fmol of D4676, DM4676, LD4676 or LDM4676 in buffer containing 10 mM HEPES, pH 7.2, and 20 mM KCl. The gels were dried, exposed to a storage phosphor screen and visualized using a Typhoon Trio scanner. In vitro RNase H assay Target RNA, consisting of 269 nt from the 5′ end of the HCV genome and the region spanning positions 3081 to 5943, was synthesized in vitro using an mMESSAGE mMACHINE T7 Transcription Kit according to the manufacturer’s instructions. RNase H assays were performed as described by Kurreck and co-authors. Briefly, the reaction mixture contained 1x RNase H buffer, 0.5 U of bacterial RNase H, 5 pmol of ASO and 500 ng of FR3131 RNA. The reaction was stopped by adding 10 l of 2x stop buffer and subsequent heating to 95C for 2 min. The reaction products were separated on a 0.8% TAE agarose gel and detected with ethidium bromide staining. The kinetics of ASO:RNA duplex cleavage were analyzed using pre-formed ASO:RNA duplexes. Briefly, 1 fmol of the labeled duplexes was mixed with 0.05 U of RNase H in 1x RNase H buffer, and the reaction was performed at 37C. Aliquots were col