Figure 4.

Gene silencing by 2′-azido-2′-deoxyuridine- and 2′-azido-2′-deoxyadenosine-modified siRNA. A) Sequence of the BASP1 siRNA used in this study;[21a] nucleosides in red indicate positions of 2′-azido modification. B) Biological activities of 2′-azido siRNAs directed against BASP1 mRNA. Chicken DF-1 cells grown on 60 mm dishes were transiently nucleofected with 0.12 nmol (∼1.5 μg) aliquots of unmodified commercial (Qiagen) or own (unmod) synthetic siRNAs (SIR) directed against the chicken BASP1 mRNA, or of siRNAs containing azido (Az) groups at the indicated nucleotides 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 two days after siRNA delivery and 5 μg-aliquots were analyzed by Northern hybridization by using DNA probes specific for the chicken BASP1 gene, or the quail housekeeping GAPDH gene. RNAs (5 μg) from normal quail embryo fibroblasts (QEF), and from QEF transfected with the retroviral construct RCAS-v-myc[21b] encoding the BASP1-suppressing v-Myc oncoprotein were used as controls. The electrophoretic positions of ribosomal RNA are indicated in the margin. All siRNAs depicted contain overhangs of 2′-deoxynucleosides (lower case letters). SIR random: 5′-UCUGGGUCUAAGCCAAACAUT/5′-UGUUUGGCUUAGACCCAGAUdG.