Figure 8

Importance of various sequence motifs for small interfering (si) RNA off-target effects on transforming growth factor-β receptor 2 (TGFBR2). Percentage of siRNAs with certain sequence motifs found in the TGFBR2 3' untranslated region (UTR) are shown for the TGFBR2 siRNA hits (dark blue bars) and the non-hit population (light blue bars). Higher blue bars indicate that a prediction based on this feature would be more successful at predicting off-target effects, and the strength of such a pattern is measured by the enrichment factor r, which is the ratio of the frequency of a given sequence motif in the seed regions of TGFBR2 siRNA hits (dark blue) divided by the frequency of that motif in the seed regions of non-siRNA hits (light blue). (A) Multiple heptamers, although rare, were more potent at silencing TGFBR2. (B) The occurrence of an A in the target TGFBR2 UTR opposite position 1 of the siRNA significantly increased the potency of the siRNA (9.2 > 3.4). (C) The occurrence of an A or U opposite position 9 of the siRNA or a match at position 9 increased the potency of an siRNA. (D) 3' pairing, in particular at position 16, seemed to increase siRNA off-target potency. For three-mer and four-mer binding, an offset of no more than two nucleotides was allowed, but the position 16 match was to position 16 of the target.