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 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.
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:
- Sense strand is modified to prevent interaction with RISC and favor antisense strand uptake
- Antisense strand seed region is modified to minimize seed-related off-targeting
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.