Figure 1.
Analysis in Drosophila Embryo Lysate of a Tiled Set of siRNAs Targeting Human SOD1
(A) Sequences of the guide siRNA strands used, indicating the site of the G:G mismatch. The third nucleotide from the 3′ end of the sense strand of the siRNA (not shown) was mismatched with the guide, creating an unpaired 5′ end to facilitate entry of the guide strand into RISC. For example, where the first nucleotide of the guide strand was U, the sense strand was changed to C, and vice versa. Where the first guide nucleotide was G, the corresponding sense strand was designed to be A, and vice versa. The mutant (matched) and wild-type (mismatched) SOD1 mRNA sequences targeted by the siRNAs are shown below.
(B) Rate of target cleavage in Drosophila embryo lysate of mutant (filled circle) or wild-type target (open circle) for each siRNA in the tiled set.
(C) Comparison of a 5′ paired and a 5′ unpaired P11 siRNA (B) reveals that the 5′ paired siRNA exaggerates the inherent discrimination of this siRNA between matched and mismatched target RNAs.
Figure 2.
Mismatched Target Cleavage after 24-h Incubation in Drosophila Embryo Lysate
To detect low levels of target cleavage, long-time scale reactions were performed with the concentration of RISC greater than that of the RNA target for those siRNAs (P5, P9, P10, P12, P13, P14, P15, P16, and P19) that showed high levels of discrimination between the matched and mismatched targets in Figure 1.
Figure 3.
Analysis of Tiled siRNAs against an SOD1-Luciferase Fusion in Cultured Human Cells
(A) Relative firefly luciferase expression for siRNAs (Figure 1A) co-transfected at either 2 nM (gray bars) or 20 nM (white bars) with a reporter plasmid containing the mutant (matched) SOD1 sequence fused to the luciferase coding sequence.
(B) The same set of siRNAs was analyzed by co-transfection with a reporter plasmid containing the wild-type (mismatched) SOD1 sequence, creating a G:G clash, or (C) a reporter plasmid encoding a U at the same position, creating a G:U wobble.
Each experiment was performed in triplicate and the data were normalized to luciferase expression measured using a GFP siRNA. Average ± standard deviation is shown.
Figure 4.
Microarray Analysis of 5′ Unpaired SOD1 siRNAs
siRNAs from Figure 1A were transfected at 100 nM into HeLa cells and total cellular RNA isolated 24 h later.
(A) Microarray analysis of genes down-regulated by siRNAs. Gene expression profiles were determined by competitive hybridization of amplified mRNA from siRNA-treated versus mock-treated cells. Shown is a heat map depiction of mRNAs whose expression decreased following siRNA transfection (p < 0.01, in one or two experiments, except for SOD1). Regulated genes are in columns, experiments in rows. The experiment using P17 was lost. Teal, genes having decreased mRNA levels compared to mock transfected cells, magenta, genes having increased mRNA levels. The color bar indicates log10 expression ratio transfected/mock transfected cells, −0.6 (teal) to +0.6 (magenta; i.e., 4-fold). Genes in yellow boxes are representative groups of transcripts enriched for seed region hexamer matches (see Table 1). The arrow indicates the position of the wild-type SOD1 mRNA, compared to a mock HeLa cell transfection.
(B) Endogenous SOD1 mRNA levels were determined by quantitative RT-PCR for each siRNA transfection. Shown is the mean ± standard deviation of three replicate determinations for each siRNA. Endogenous wild-type SOD1 mRNA is mismatched to the siRNAs used; taller bars imply greater discrimination against the mismatched target RNA.
Table 1.
Off-Target Analysis Shows that Most of the SOD1 siRNAs Designed to Be Functionally Asymmetric Load the Guide Strand into RISC
Figure 5.
Purine:Purine Mismatches Provide the Greatest Discrimination for mRNAs Differing at a Single Nucleotide
(A) The asymmetry of a fully base-paired P10 siRNA was measured using a firefly luciferase reporter containing sense or antisense SOD1 sequences. Even at high concentrations of siRNA, only the sense target (open squares) was efficiently silenced, compared to the antisense target (filled squares). Thus, the guide strand of the P10 siRNA was predominantly the antisense strand, consistent with the off-target analysis in Table 1.
(B) All possible single nucleotide pairs were examined for the P10 siRNA. Among the perfectly complementary siRNA:mRNA pairs, a position 10 G:C pair triggered greater silencing than an A:U pair. Purine:pyrimidine and pyrimidine:pyrimidine mismatches displayed intermediate levels of silencing. The least silencing, i.e., greatest discrimination, was observed with purine:purine mismatches.
(C) Over a range of concentrations, the pyrimidine:purine mismatches show moderate levels of discrimination compared to the perfectly matched siRNA:mRNA pair. U:C mismatches, triangles; U:U mismatches, diamonds; U:G mismatches, circles; U:A matched pair, squares.
(D) Purine:purine mismatches cannot be overcome by high concentration of siRNAs and therefore the observed discrimination is not an artefact of low concentration. A:G mismatches, circles; A:A mismatches, squares; A:C mismatches, triangles; A:U matched pair, diamonds. Mismatches are reported as siRNA nucleotide:target RNA nucleotide.
Figure 6.
Purine:Purine Mismatches Are Tolerated at Some, but Not Other, siRNA Positions
(A) Purine residues were placed at each position, N1 to N19, along the P10 siRNA. Targets were constructed so that siRNA:mRNA target pairs would result in a purine:purine mismatch. Luciferase activity was measured for each pair.
(B) The P4 siRNA (Figure 1A) was used as a scaffold for the analysis of the effect of purine:purine mismatches on reporter silencing. Taller bars correspond to greater single nucleotide discrimination.
Table 2.
Mismatches at Position 16 Confer a High Level of Single Nucleotide Discrimination