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The patented ON-TARGETplus modification pattern for specificity, combined with the SMARTselection algorithm for efficient, guaranteed target gene silencing, makes ON-TARGETplus siRNA the premium choice for optimal knockdown and reduced off-targets.
ON-TARGETplus SMARTpool and individual siRNAs are available as pre-designed, genome-wide reagents for human, mouse and rat. Simply search for your gene of interest and choose from the available product formats and quantities.
Dharmacon scientists and collaborators demonstrated in 2006 (see Reference 1) that siRNA off-targets are mediated by antisense seed-region interactions, initiating the development of a dual-strand modification pattern:
A landmark publication (see Reference 2) from the Dharmacon research group was the first to experimentally demonstrate the key role of the seed region in mediating off-targets. A subsequent 2008 paper (see Reference 3) showed the importance of seed frequency in the 3'UTR as in indicator of its likelihood to cause off-targets. These principles were subsequently applied to our siRNA designs to improve specificity:
ON-TARGETplus reagents are routinely used at 5 to 25 nM concentration. The calculations below, based on 25 nM, are for estimation purposes only and assume no loss from pipetting.
siGENOME and ON-TARGETplus siRNA reagents (SMARTpool and three of four individual siRNAs) are guaranteed to silence target gene expression by at least 75% at the mRNA level when used under optimal delivery conditions (confirmed using validated positive control and measured at the mRNA level 24 to 48 hours after transfection using 100 nM siRNA).
Note: Most siGENOME and ON-TARGETplus siRNA products are highly functional at 5 to 25 nM working concentration.
The effect of silencing ARPC1B on cell migration was studied in a breast cancer cell line. A monolayer of cells was uniformly scraped and the rate of cell migration to close the scrape (wound healing) was evaluated. Both unmodified and ON-TARGETplus siRNA reagents induced potent target knockdown. Inconsistent phenotypes due to off-target effects (red outline), were observed for cells transfected with unmodified individual siRNAs. The unmodified SMARTpool improved the false phenotype considerably, while the ON-TARGETplus SMARTpool significantly reduced off-target effects to produce a consistent phenotype.
In collaboration with Kaylene Simpson, Laura Selfors and Joan Brugge, Harvard Medical School.
Panels (A) and (B) are representative examples of off-target signatures with and without application of ON-TARGETplus modifications to (A) a single siRNA and (B) a SMARTpool reagent. Green bars indicate genes with 2-fold or more reduction of expression when treated with the indicated siRNA reagent.The ON-TARGETplus modifications reduced the off-targets when compared to unmodified siRNA. Pooling of siRNA and the ON-TARGETplus modification pattern independently, and in combination, provide significant reduction in off-target gene silencing. Panel (C) represents quantitation of off-targets (down-regulated by 2-fold or more) induced by the indicated siRNA reagents targeting 10 different genes (4 siRNAs per gene or a single SMARTpool reagent). Off-targets were quantified using microarray analysis (Agilent) then compiled. Each shaded box represents the middle 50% of the data set. Horizontal line in box: Median value of the data set. Vertical bars: minimum and maximum data values.
The ON-TARGETplus dual-strand chemical modification begins with the sense (passenger) strand being blocked from RISC uptake to favor antisense (guide) strand loading and reduce passenger strand-induced off-targets. However, the majority of siRNA off-targets are driven by the seed region of the guide strand. ON-TARGETplus is modified within its seed region to destabilize miRNA-like activity and improve specificity to the desired target for potent knockdown.
ON-TARGETplus siRNA designs leverage sophisticated bioinformatics to reduce the likelihood of miRNA-like off-targets from high-frequency or highly conserved miRNA seed regions. siRNAs with low seed frequency have a significantly lower number of off-targets than siRNAs with medium or high frequency seeds. Five siRNAs with low, medium, or high frequency seed regions were transfected into HeLa cells and their associated off-target signatures assessed via global expression profiling (Agilent 22K platform). siRNA sequences were constant at positions 1 and 8-19, only the seed regions (positions 2-7) were altered.
Low frequency seeds: < 350 occurrences in the HeLa transcriptome
Medium frequency: 2500-2800 occurrences
High frequency: >3800 occurrences
A 2006 publication demonstrates that off-target effects are primarily driven by antisense strand seed activity†. Therefore, sense strand inactivation alone does not decrease the total number of off-target genes.
ON-TARGETplus modifications account for both strands:
The ON-TARGETplus modification pattern dramatically reduces off-targets. Off-target effects induced by the indicated siRNAs were quantified using microarray analysis. For each target, three different siRNAs were used: unmodified, sense strand-inactivated, and ON-TARGETplus-modified. Data shown represents genes down-regulated by two-fold or more. HEK293 cells were transfected with 100 nM siRNA using 0.2 μL of DharmaFECT 1. Data was analyzed at 24 hours.
A. L. Jackson et al., Position-specific chemical modification increases specificity of siRNA-mediated gene silencing. RNA. 12(7), 1197-1205 (July 2006).
A. Birmingham et al., 3'-UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods. 3(3), 199-204 (March 2006).
E. M. Anderson et al., Experimental validation of the importance of seed complement frequency to siRNA specificity. RNA. 14(5), 853-861 (May 2008).