C911: A bench-level control for sequence specific siRNA off-target effects

Abstract

Small interfering RNAs (siRNAs) have become a ubiquitous experimental tool for down-regulating mRNAs. Unfortunately, off-target effects are a significant source of false positives in siRNA experiments and an effective control for them has not previously been identified. We introduce two methods of mismatched siRNA design for negative controls based on changing bases in the middle of the siRNA to their complement bases. To test these controls, a test set of 20 highly active siRNAs (10 true positives and 10 false positives) was identified from a genome-wide screen performed in a cell-line expressing a simple, constitutively expressed luciferase reporter. Three controls were then synthesized for each of these 20 siRNAs, the first two using the proposed mismatch design methods and the third being a simple random permutation of the sequence (scrambled siRNA). When tested in the original assay, the scrambled siRNAs showed significantly reduced activity in comparison to the original siRNAs, regardless of whether they had been identified as true or false positives, indicating that they have little utility as experimental controls. In contrast, one of the proposed mismatch design methods, dubbed C911 because bases 9 through 11 of the siRNA are replaced with their complement, was able to completely distinguish between the two groups. False positives due to off-target effects maintained most of their activity when the C911 mismatch control was tested, whereas true positives whose phenotype was due to on-target effects lost most or all of their activity when the C911 mismatch was tested. The ability of control siRNAs to distinguish between true and false positives, if widely adopted, could reduce erroneous results being reported in the literature and save research dollars spent on expensive follow-up experiments.

Figure 1. On and off-target effects of siRNAs and their controls.

An siRNA (A, left panel) consisting of two complementary 19-mers of RNA (with two-base overhangs) is divided here conceptually into the 5′ end of the anti-sense strand (teal) the middle of the siRNA (black) and the 3′ end of the anti-sense strand (red). siRNAs are designed to be the reverse-completment of the mRNA sequence they are targeted to down-regulate (A, middle panel), but matches of the seed sequence of an siRNA to the 3′UTR of other mRNAs can result in their off-target down-regulation as well (A, right panel). A scrambled siRNA (B) eliminates the match to the target mRNA and thus will not down-regulate it, but also eliminates the off-target effects due to matches to the seed sequence (while, perhaps, creating new off-target effects against the new seed sequence). The C911 mismatch control (C) reduces or eliminates the down-regulation of the targeted mRNA by taking the complement of the middle three bases (green), but maintains the off-target effects of the original siRNA by keeping anti-sense and sense strand seed sequences intact. In this manner, comparison of effects elicited by the original siRNA and the C911 mismatch control should allow us to distinguish phenotypes that are due to down-regulation of the intended target rather than off-target effects.

Figure 2. Examples selected for test set.

Common seed analysis plots for two genes which had an siRNA showing significant knockdown (greater than 2-fold decrease in luciferase signal in comparison to non-silencing control). The dashed line represents the median response for the whole genome library. (A) POLR2A was selected as a true positive because multiple siRNAs generated the same phenotype, the role of RNA polymerase in transcription is already established, and siRNAs containing the same seed sequences (grey circles) did not show a trend towards inhibition of the reporter. (B) In contrast, PLXDC1 was selected as a false positive because it has no known role in transcription, other siRNAs against the same gene fail to elicit the same phenotype, and there is an obvious trend of down-regulating the reporter for all other siRNAs containing the same hexamer (GAGTAG) or heptamer seed sequence.

Figure 3. Results for three different classes of negative control.

siRNAs which downregulate a luciferase reporter more than two-fold (teal, panels A,B, and C) were selected and categorized as either true or false positives based on biological annotation, the activity of other siRNAs against the same gene, and observed activity of other siRNAs with the same seed sequence. Three types of negative control were then tested for their ability to distinguish true positives from false positives. As expected, scrambled versions of the siRNAs (red, panel A) reduced or eliminated their activity, regardless of whether it was on (true positive) or off-target (false positive). In contrast, the C911 mismatch design (green, panel B) showed a large reduction in activity for only true positive siRNAs, indicating that changing bases 9–11 of the siRNAs to their complement successfully disrupted on-target activity while maintaining off-target activity. The C10 mismatch design (yellow, panel C) also maintained off-target effects for false positives, but in some cases failed to reduce the on-target phenotype observed in the true positive group as drastically as the C911 mismatch. Statistically significant p-values for the t-test are marked by *** (p-value < = .001), ** (p-value < = .01), and *(p-value < = .05). The dashed line represents the median response for the whole genome library. Error bars represent the calculated standard error (standard deviation divided by the square root of the number of observations).