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, 19 (9), 1676-87

Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-Target Silencing

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Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-Target Silencing

Pooja Dua et al. Mol Ther.

Abstract

Off-target gene silencing is a major concern when using RNA interference. Imperfect pairing of the antisense strand with unintended mRNA targets is one of the main causes of small interfering RNA (siRNA) off-target silencing. To overcome this, we have developed "bulge-siRNA," a modified siRNA backbone structure with a single nucleotide (nt) bulge placed in the antisense strand. We found that siRNAs with a bulge at position 2 of the antisense strand were able to discriminate better between perfectly matched and mismatched targets, with no loss in silencing of the intended target. Genome-wide analysis also revealed that the bulge-siRNAs significantly reduced off-target silencing of transcripts with complementarity to the seed region of the siRNA antisense strand. When compared to 2'-methoxy ribosyl (2'-OMe) modified siRNAs previously developed to alleviate antisense off-target silencing; the bulge modification could better discriminate between on- versus off-targets. Our results suggest that the bulge-siRNA structure is a simple, yet superior alternative to chemical modifications for minimizing off-target silencing triggered by conventional siRNA structures.

Figures

Figure 1
Figure 1
On-target silencing activity of bulge-small interfering RNA (siRNA) depends upon position of bulge. (a) The sequence and structure of siRNAs targeting survivin mRNA and the corresponding hypothetical structures. The number in the siRNA nomenclature represents the position of the single nucleotide bulge (gray) from the 5′-end of the antisense sequence. (b) Bulge-modified siSurvivin show position-dependent silencing activity. HeLa cells were transfected with 10 nmol/l of siRNA targeting GFP expression (siGFP) or siSurvivin siRNAs. Results from real-time quantitative PCR (qPCR) are shown as mean ± SE from three independent experiments.
Figure 2
Figure 2
Bulge modification at position 2 shows significant discrimination between perfectly matched and mismatched antisense targets. (a) Schematic view of pMIR lucifersase reporter used to monitor small interfering RNA (siRNA) on-target and off-target silencing. A single copy of the antisense target site, wild-type (WT) or mismatch/Mut was cloned in the 3′-untranslated region (UTR) of the Firefly luciferase reporter to generate an on-target or off-target reporter, respectively. Drawings are not to scale. (b) On-target and off-target silencing of unmodified and bulge-modified siSurvivin. HeLa cells were co-transfected with on-target or off-target pMIR reporter plasmid and 10 nmol/l of siSurvivin constructs for 24 hours. Renilla luciferase vector was transfected as an endogenous control to normalize differences in cell number. After 24 hours of transfection, Firefly luciferase signals were normalized with Renilla luciferase signals. The normalized luciferase intensity relative to siRNA targeting GFP expression (siGFP) (shown as 100%) is given here. The position and type of mismatch formed are indicated on the x axis. (c) Fold discrimination between on- and off-target silencing. Ratio of luciferase activities for the mismatched target versus wild-type target was calculated as fold discrimination and average of discrimination for all the targets is shown for each siRNA. Results are represented as mean± SE of three independent experiments.
Figure 3
Figure 3
Bulge-siSurvivin is superior to 2′-methoxy ribosyl (2′-OMe) modified siSurvivin in off-target discrimination and reduction in off-target silencing is independent of the identity of the bulge nucleotide. (a) Sequence of unmodified and modified siSurvivin constructs with bulge and 2′-OMe modifications at position 2. Bulge and 2′-OMe modified nucleotides are marked in gray, respectively. (b) IC50 values of unmodified and modified siSurvivin were determined by real-time quantitative PCR (qPCR). (c) On-target and off-target silencing of siSurvivin constructs at 10 nM concentrations. HeLa cells were co-transfected with on-target or off-target pMIR reporter plasmid and 10 nM of siSurvivin constructs for 24 hr. The Firefly to Renilla normalized luciferase intensity relative to the siRNA targeting GFP expression (siGFP) is given here. (d) Fold discrimination between on- and off-target silencing. Ratio of luciferase activities for the mismatched versus wild-type target was calculated as fold discrimination and the average discrimination of both the mismatched targets is shown for each siRNA at three different concentrations. Results are shown as mean ± SE of three independent experiments.
Figure 4
Figure 4
Bulge modification reduces off-target silencing of siMAPK. (a) Sequences of unmodified and modified siMAPK-14 with bulge and 2′-OMe modifications at position 2 of antisense strand. (b) IC50 values of MAPK14 small interfering RNAs (siRNAs). (c) On-target and off-target silencing of siMAPK-14 constructs at 10 nmol/l concentrations. (d) Average of fold discrimination between on- and off- target silencing of both the mismatched targets is shown for each siRNA at three different concentrations. For details see descriptions of Figure 3.
Figure 5
Figure 5
Bulge modification enhances on-target silencing activity of siMPHOSPH1 but reduces off-target silencing. (a) Sequences of unmodified and modified siMPHOSPH1 with bulge and 2′-methoxy ribosyl (2′-OMe) modifications at position 2 of the antisense strand. (b) IC50 values of MPHOSPH1 small interfering RNAs (siRNAs). (c) On-target and off-target silencing of siMPHOSPH1 constructs at 10 nmol/l concentrations. (d) Average of fold discrimination between on- and off-target silencing of both the mismatched targets is shown for each siRNA at three different concentrations. For details see descriptions of Figure 3.
Figure 6
Figure 6
Bulge modification reduces small interfering RNA (siRNA)-mediated cellular toxicity. Cells were transfected with 10 nmol/l siRNAs. After 4 days, cell viability was determined by MTT assay, considering lipofectamine transfection control as 100% viable. The silencing efficiency of the siRNAs at 10 nmol/l concentration was calculated by real-time quantitative PCR and a correlation of siRNA activity to cell viability is shown. Results are represented as mean ± SE of three independent experiments.
Figure 7
Figure 7
Microarray-based genome-wide off-target profiling of siSurvivin. MA plots showing the changes in the expression of transcripts with antisense seed match (red dots) or no canonical matches in their 3′-UTR (represented as others, gray dots) upon siRNA transfection. (a) siSurvivin, (b) siSurvivin-2′C′, (c) siSurvivin-2′OMe. Transcripts with homology to new seed generated by the bulge are also shown (modified antisense, yellow dots). Cumulative distribution of antisense, sense, or modified seed-matched transcripts in comparison to transcripts with no seed matches are shown as log2 of fold change in expression upon treatment with (d) siSurvivin, (e) siSurvivin-2′C′, and (f) siSurvivin-2′OMe. (g) Number of transcripts with respective seed homology downregulated by 50% or more. (h) Reduction in siSurvivin-mediated off-target silencing by bulge or 2′-OMe modifications. Antisense seed-matched transcripts that were downregulated by more than twofold upon siSurvivin treatment (siSurvivin off-targets) were collected and average inhibition in their expression was calculated in siSurvivin, bulge, and 2′-OMe-modified constructs. The reduction in the silencing of these off-targets upon bulge or 2′-OMe modifications is shown.
Figure 8
Figure 8
5′-RACE assay shows similar cleavage products for unmodified and bulge-modified MAPK14 small interfering RNA (siRNAs). PCR products of 5′-RACE assay from siMAPK14 transfected cells. The mRNA cleavage sites were analyzed by 5′-RACE assay and sequencing. The MAPK14 target mRNA (upper strand) sequence with the antisense strand (lower) are given. The cleavage sites are marked with arrowheads and the numbers show the frequency of clones sequenced.

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