Molecular basis for improved gene silencing by Dicer substrate interfering RNA compared with other siRNA variants

Abstract

The canonical exogenous trigger of RNA interference (RNAi) in mammals is small interfering RNA (siRNA). One promising application of RNAi is siRNA-based therapeutics, and therefore the optimization of siRNA efficacy is an important consideration. To reduce unfavorable properties of canonical 21mer siRNAs, structural and chemical variations to canonical siRNA have been reported. Several of these siRNA variants demonstrate increased potency in downstream readout-based assays, but the molecular mechanism underlying the increased potency is not clear. Here, we tested the performance of canonical siRNAs and several sequence-matched variants in parallel in gene silencing, RNA-induced silencing complex (RISC) assembly, stability and Argonaute (Ago) loading assays. The commonly used 19mer with two deoxythymidine overhangs (19merTT) variant performed similarly to canonical 21mer siRNA. A shorter 16mer variant (16merTT) did not perform comparably in our assays. Dicer substrate interfering RNA (dsiRNA) demonstrated better gene silencing by the guide strand (target complementary strand), better RISC assembly, persistence of the guide strand and relatively more loading of the guide strand into Ago. Hence, we demonstrate the advantageous properties of dsiRNAs at upstream, intermediate and downstream molecular steps of the RNAi pathway.

Figure 1.

Design of sequence-matched siRNA variants used in this study. (a) H5 series of variants. (b) H6 series of variants. DNA nucleotides are represented as underlined ‘d_’. The predicted in vitro Dicer cleavage sites are indicated by the open arrowheads. All variants are sequence-matched with respect to the 5′-end of the 21mer bottom strand. The color scheme used here will be consistent throughout the remainder of this report.

Figure 2.

Gene-silencing activity of siRNA variants. Gene silencing was assessed by dual luciferase assay using plasmids that report the RNAi activity of the bottom (blue and green) or top (red and orange) strands for the H5 (a) and H6 (b) set of variants. Assay performed in HCT116 cells. All data points were normalized to an equimolar amount of non-targeting siRNA. n ≥ 2, in duplicate, mean ± SEM. IC50 values for each strand of each siRNA variant are included as insets.

Figure 3.

Robust high-molecular-weight complex formation by dsiRNAs in RISC assembly assays. Immunoblotting confirmed the transient expression of epitope-tagged RNAi proteins (top). ‘non’ = cells without respective plasmid transfection. EMSA assay performed with the H5 (a) or H6 (b) sets of variants. Asterisk (*) indicates position of the 5′ ³²P label. Labeled RNAs in the absence of cell lysate (‘−’) serve as negative controls. Amount of band shift was calculated as the sum of shifted bands divided by the respective negative control lane. Images and densitometry are representative of two biological replicates.

Figure 4.

Synthetic dsiRNA products not similar to dsiRNA in gene silencing. (a and b) Schematic of H5 20 + 2mer and H6 19 + 3mer siRNA variants, compared with their sequence-matched 21mer and dsiRNA format. Boxed nucleotides represent nucleotides not predicted by in vitro Dicer processing. (c and d) Dual luciferase gene-silencing assay. n = 3, in duplicate, mean ± SEM. P-value shown from unpaired Student’s t-test.

Figure 5.

More bottom strands load into Ago2 for dsiRNAs compared with canonical siRNAs. Experiment performed in HEK293FLAGHA_Ago2 cells. Immunoblotting confirmed the IP procedure for the H5 (a) and H6 (b) set of variants, respectively. Lysate from wild-type HEK293 cells transiently transfected with pFLAG-Ago2 or pFLAG empty vector serve as a positive and negative control, respectively. Northern blots showing the levels of input and immunoprecipitated small RNAs for the H5 (c) and H6 (d) set of variants. Non-transfected cells or cells transfected with siGFP served as negative controls. White arrows indicate full-length (not Dicer processed) dsiRNA strands. Representative images from three independent biological replicates.

Figure 6.

Loading of each strand of siRNA variants into Ago2 is not dependent on mRNA target presence. Co-transfections of the indicated psiCHECK strand sensor (scaling up from the amount used in Figure 2) and 10 nM of the indicated H5 (a) or H6 (b) siRNA variant were transfected into HEK293FLAGHA_Ago2 cells. Small RNA immunoprecipitation assay followed by Northern blotting was conducted as in Figure 5. Cells treated with siGFP served as negative controls. Additional size markers (lanes 1–4) are included to indicate the size of the probed small RNA species.