Figure 5.

Gene silencing by 2′-SCF₃-modified siRNAs. A) Sequence of the brain acid soluble protein 1 gene (BASP1)[46] targeting siRNA duplex used in this study; nucleosides in red indicate positions for 2′-SCF₃ modification tested. B) Biological activities of 2′-SCF₃-adenosine or 2′-SCF₃-guanosine modified siRNAs directed against BASP1 mRNA. Chicken DF-1 cells grown on 60 mm dishes were transiently nucleofected with 0.24 nmol (∼3.0 μg) aliquots of unmodified (SIR) or modified siRNAs (SIR 2′-SCF₃) on sense (s) or antisense (as) strands. An equal aliquot of siRNA with a shuffled (random) nucleotide sequence was used as a control. Total RNAs were isolated 2 days (left panel) or 3 days (right panel) after siRNA delivery, and 5 μg aliquots were analyzed by Northern hybridization using a DNA probe specific for the chicken BASP1 gene, and subsequently a probe specific for the housekeeping quail GAPDH gene.[46] The levels [%] of BASP1 expression (Expr.) were determined using a phosphorimager and are depicted as bars relative to mock transfections (no SIR, 100 %). The electrophoretic positions of rRNAs are indicated in the margin. SIR random: 5′-UCUGGGUCUAAGCCAAACAUT/5′-UGUUUGGCUUAGACCCAGAUdG.