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

<p>An siRNA (<b>A, left panel</b>) 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 (<b>A</b>, <b>middle panel</b>), 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 (<b>A</b>, <b>right panel</b>). A scrambled siRNA (<b>B</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 (<b>C</b>) 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.