FIGURE 3.

shRNA processing and silencing efficiency is overhang dependent. (A) Diagrams depicting the various 5′ and 3′ overhangs tested on identical shRNA stem–loops. The 5′-27 nt (Silva et al. 2005) and 5′-XbaI (Boden et al. 2004) variants were used in prior shRNA and miRNA comparison studies. (B) Plasmids expressing the shRNA variants were transfected into HEK293 cells, and small transcript Northern blot (probing for antisense [AS] or sense [S] sequences) with densitometry analysis (values for AS and S bands are shown below blots) was performed 48 h later to assess shRNA processing efficiency (n = 3). Results show that 5′ overhang variants yield less precursor (Pre-) and antisense (AS) RNAs compared to the optimized shRNAs with U_2–4 3′ overhangs (derived from Pol-III transcription termination (Ng et al. 1979; Ohshima et al. 1981; Kunkel et al. 1986). Appropriate strand loading was observed for each shRNA variant (i.e., AS:Pre->S:Pre-). (C) Plasmids expressing the shRNA variants were transfected into HEK293 cells, and nuclear/cytoplasmic fractionation and RNA isolation was performed 24 h later. Equal amounts of nuclear and cytoplasmic RNAs for each treatment group were analyzed by small transcript Northern blot (probing for antisense sequences) to evaluate where precursor (Pre-) shRNAs accumulate. Results demonstrate that the two most highly expressed shRNAs (3′-U_2–4 or 3′-CU_2–4) accumulate in both nuclear and cytoplasmic fractions, suggesting that saturation of both Exportin-5 and Dicer may occur. Conversely, buildup of the other shRNA variants was present primarily in the nucleus, suggesting lack of export due to suboptimal overhangs. Note: the presumed Drosha-cleavage product of the 5′-27 nt shRNA is exported and accumulates slightly in both compartments. The blot was stripped and reprobed for the nuclear U6 splicing RNA to control for fractionation and loading. Nonspecific bands (NS) present following the control probing are shown as additional fractionation and loading controls. (D) Silencing of intended (solid bars) or unintended (empty bars) target strands was assessed by cotransfection of shRNA-variant and RNAi luciferase reporter expression plasmids into HEK293 cells, and Dual Glo Luciferase assays were performed at 24 h. Results are shown as mean±SD (n = 3) relative to mock-treated controls and confirm that suboptimal overhangs decrease silencing efficiency. Notably, each shRNA preferentially silenced the intended target (transfected at 1:20 RNAi:target) relative to the unintended target (transfected at 3:1 RNAi:target).