Dicer cleavage of sshRNAs in their dimeric forms. sshRNAs with (*) or without heating and snap-cooling were subjected to recombinant Dicer treatment as indicated. Samples were analyzed by 10% nondenaturing PAGE with SYBR Gold staining. (A) sh1 (25-bp stem; positive control) and SG119. (B) SG 142, SG112, and SG113.

Target mRNA cleavage in vivo. 5′-RACE–PCR amplification of samples with and without target-specific siRNA or sshRNA treatments was analyzed on 2% agarose gels. L, 25-bp ladder; L′, 100-bp ladder; target pSG154m alone (lane 1); SG105 (lane 2); SG119 (lane 3); SG68 (lane 4); si19-3 (lane 5); no transfection control (lane 6); and water control (lane 7).

IFN-responsive gene OAS1-induction by sshRNAs in human PBMCs. Synthetic sshRNAs (20 nM) were complexed with DOTAP (Roche) and transfected into freshly isolated human PBMC in triplicate. DOTAP alone, or DOTAP complexed with equivalent amounts of either T7 transcribed shRNA or polyI:C were used as controls. RNA was extracted from cells 24-h post-transfection and quantitative RT-PCR was performed to measure the level of GAPDH and OAS1. The RNA level of OAS1 was normalized to GAPDH and is shown relative to DOTAP alone.

Comparison of the activities of L sshRNAs against two different targets. (A) Comparison of L sshRNAs specific for the same target as siRNA si72. SG118 and SG137 have a dinucleotide UU to link the antisense and sense strands (both are 19 nt in length). SG136 has a 19-nt antisense strand directly connected with the 5′ end of a 17-nt sense strand. (B) Comparison of L sshRNAs specific for the same target as siRNA si74. All three sshRNAs have dinucleotide UU as a connection between antisense and sense strands. Values labeled “as/s” represent the nucleotide lengths of antisense and sense strands.

L sshRNA is active in monomer form. L sshRNAs were treated with or without heating (95°C for 4 min) and snap-cooling (ice/water bath for 20 min). (A) The presence of monomers and multimers was examined using native 10% polyacrylamide gel electrophoresis and SYBR Gold staining. (B) Comparison of inhibition of IRES-dependent luciferase expression with and without the heat–cool step.

Effect of single mismatches in the seed region of the sense strand of L sshRNAs on inhibitory activity. (A,B) Effect of position of mismatch. SG142-based L sshRNAs with single mismatches at positions 4–7 from the 5′ end of the sense strand were chemically synthesized and their abilities to inhibit HCV IRES-dependent luciferase expression were compared in 293FT cells. (C) Effect of a single mismatch at position 5 from the 5′ end of the sense strand on inhibition of SG117-based sshRNAs (s/as = 18/18), compared with the fully matched SG117 and si19-3.

Comparison of L sshRNA activity with various loop structures and base pairs adjacent to the loop. (A) Loop length comparison, using L sshRNAs against the same target region as sh68 and si19-3, but with various lengths and sequences of loops. sshRNAs were chemically synthesized and their abilities to inhibit HCV IRES-dependent luciferase expression were compared in 293FT cells. (B) Loop-adjacent base-pair comparison. sshRNAs against the same target region as si72, but with loops of 5 nt (SG72 and SG72L) and 2 nt (SG118 and SG103) and left-loop (SG72L and SG118) and right-loop (SG72 and SG103) orientations were compared for their inhibitor activity in 293FT cells. Loop sequences are underlined.

sshRNAs monomers are not Dicer substrates. Eight picomoles of each synthetic shRNA (after heating and snap-cooling) were incubated in a 10 μL reaction in the presence of 1 U of recombinant Dicer enzyme (Stratagene) and buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 8), and 2.5 mM MgCl₂ for 18 h at 37°C. Control reactions that contained each shRNA but lacked Dicer were incubated in parallel. Samples were analyzed by (A) nondenaturing 10% PAGE and (B) denaturing 12% PAGE (8 M urea/20% formamide) and were stained with SYBR Gold. Note that SYBR Gold did not stain single-strand RNA as strongly as double-strand RNA.

Comparison of the activities of L sshRNAs differing in 3′ overhang and stem length. (A) Overhang comparison. L sshRNAs against the same target region as si19-3 and SG142, but with or without a 3′ overhang were chemically synthesized and their ability to inhibit HCV IRES-dependent luciferase expression was compared in 293FT cells. (B) Stem length comparison. L sshRNAs (UU-loop) against the same target region as SG142 and si19-3, but with different stem lengths were compared for their inhibitory activity in 293FT cells. SG105 differs from SG142 in lacking a 3′-UU overhang. (C) SG119 and SG117 comparison in 293FT cells. SG119 has a 19-nt antisense strand directly linked with a 17-nt sense strand, probably forming a 16-bp duplex and 4-nt loop (UGCA). (D) Stem length comparison with SG119 derivatives. SG131 and SG132 have UU to connect the 3′ end of the antisense and the 5′ end of the sense strands. They were compared with SG119 for target knockdown in 293FT cells. si131 has two complementary strands 16 nt in length and UU overhangs at the 3′ ends. Values labeled “as/s” represent the nucleotide lengths of antisense and sense strands.

Structure and activity of R and L sshRNAs. (A) General structures of R and L sshRNAs. (B) Representative structures of L sshRNAs, with sense and antisense strands of equal or unequal length. (C) Schematic representation of luciferase reporter plasmids expressing the target for si/shRNAs. The IRES/fLuc plasmid has the near full-length HCV IRES upstream of the firefly luciferase open reading frame. The expression of the firefly luciferase is driven by the HCV IRES. The psiCHECK-2-pIRES plasmid was constructed based on psiCHECK-2 from Promega. The targeting regions (part of the HCV IRES [pIRES]) of si19-3 (black line), si72 (light gray line), and si74 (dark gray line) were individually inserted at the 3′ end of hRluc by site-directed mutagenesis. (D) Comparison of the activities of L and R sshRNAs using IRES/fLuc plasmid in 293FT cells. sshRNAs against the same target were chemically synthesized with right- and left-hand loops (SG68 and SG68L). These shRNAs were cotransfected in triplicate into 293FT cells with IRES-fLuc plasmid. The luciferase expression was measured 48 h post-transfection. At least two independent experiments were performed and similar results were obtained. Curves were plotted using Microsoft Excel software, while IC₅₀s were calculated based on curve fitting using Erithacus GraFit software. (E) Comparison of the activities of L and R sshRNAs in Huh7 cells. Transfection conditions were as in D. SG101 was a nonspecific control for SG68 and SG68L. (F) Comparison of the activities of L sshRNA and siRNA using the dual luciferase reporter plasmid (psiCheck2-pIRES) in 293FT cells. Transfection conditions were as in D.