Figure 1

Inhibition of target gene expression by siRNAs expressed from the TAR microRNA backbone. (A) siRNA sequence targeting the earlier reported site II of HIV rev was folded in silico and the configurations that retained the correct Tat binding region were selected for further studies. Configurations I & II have the lower single nucleotide bulge removed or both the bulges retained respectively. Configuration III is the anti-site II Rev shRNA with the 9 nt loop reported earlier by us[28]. Arrows indicate single nucleotide bulge (B) Target site corresponding to the site II of HIV rev is cloned in the 3' untranslated region of the renilla luciferase ORF in the siCHECK vector. The three configurations of siRNA were invitro transcribed and treated with Calf intestinal alkaline phosphatase. The CIP treated RNA were then cotransfected with the psiCHECK plasmid having the Rev Target site. Dramatic inhibition of reporter gene expression is observed with all three configurations. The configuration where the lower bulge is removed is three-fold more potent than even the anti-Rev shRNA. (C) An siRNA targeting TGF-β is expressed in a similar fashion from the TAR miRNA backbone and co-transfected with siCHECK plasmid having the TGF-β target site. ~70% inhibition is observed with this construct suggesting that the TAR miRNA is a versatile backbone for expressing siRNA. All siCHECK assays are a mean of three experiments. CFI and CFII = Configuration I and Configuration II.