Figure 1.

agshRNAs provide efficacy, lack of passenger strand activity, and reduced competition with an endogenous miRNA compared with classical shRNAs

(A) The U6-driven RNAi effector expression cassette. Sequence and predicted secondary structure of the agsh12 and the classical sh12 with expected Ago2 and Dicer cleavage sites. The predicted guide strand seed sequence (blue) and the predicted passenger strand seed sequence (orange) are shown in bold. The 13 and S1 agshRNA-shRNA pairs are shown in Figure S1. (B) Knockdown efficacy of the U6-driven RNAi effectors in HEK293 cells measured using co-transfected dedicated dual luciferase reporters with the Renilla luciferase (Rluc) fused to a Vegfa or S1 target sequence in the sense or the AS direction (see also Figure 1D) and the firefly luciferase (Fluc) serving as an internal control for normalization. Data for each individual experiment are presented in Figures S2A–S2C. (C) The competition of the U6-driven RNAi effectors with the endogenous miR21 was investigated in HEK293 cells using a co-transfected dual luciferase reporter with a perfect miR21 target sequence. An increase in the expression of the miR21-sensitive reporter may be interpreted as reduced functional miR21 levels caused by RNAi effector-mediated competition. The H1-driven miR21-specific tough decoy (TuD21), which has previously been shown to efficiently reduce miR21-mediated silencing,²⁷ was included as a positive control, along with an empty control vector for normalization of this control. (D) Left: schematics of the Vegfa mRNA and the position of target region 12 and 13, which were fused to the Rluc of the psiCHECK dual luciferase reporters in the sense or the AS direction to create the dedicated luciferase reporters psiCHECK2-mVEGF-T12-Sense/AS or psiCHECK2-mVEGF-T13-Sense/AS. Right: schematics of the HIV-1 tat transcript with the S1 target sequence that was fused to the psiCHECK reporter in the sense or the AS direction to create psiCHECK2-S1-Sense/AS. The psiCHECK reporters also encoded Fluc. Rluc/Fluc ratio is the mean of triplicates ± SD normalized to a control. (C) A one-way ANOVA was performed followed by Šídák's multiple comparisons test between each of the agshRNAs and their corresponding shRNAs. The multiplicity-adjusted p values are reported. ∗∗∗∗p <0.0001.