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. 2006 Sep 8;2(9):e140.
doi: 10.1371/journal.pgen.0020140. Epub 2006 Jul 24.

Designing siRNA that distinguish between genes that differ by a single nucleotide

Dianne S Schwarz  1 , Hongliu Ding, Lori Kennington, Jessica T Moore, Janell Schelter, Julja Burchard, Peter S Linsley, Neil Aronin, Zuoshang Xu, Phillip D Zamore
Affiliations

Designing siRNA that distinguish between genes that differ by a single nucleotide

Dianne S Schwarz et al. PLoS Genet. .

Abstract

Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

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Conflict of interest statement

Competing interests. PDZ is a co-founder and advisor for Alnylam Pharmaceuticals, Inc., an siRNA therapeutics company.

Figures

Figure 1
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
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
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
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.
Figure 5
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
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.
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