5158 Nucleic Acids Research, 2007, Vol. 35, No. 15 Figure 2. Diﬀerent shRNA sequences compete with diﬀerent strengths against the Luc shRNA. (a) Diﬀerent concentrations of irrelevant shRNA-expressing plasmids (shL, shSII, shSI) were co-transfected with the ﬁreﬂy luciferase target (FLT) and the speciﬁc anti-luciferase shRNA (Luc). The amount used in each transfection is indicated in ng (12.5, 25, 50 or 150). The luciferase units for each sample were calculated as described in Figure 1 and in the Materials and Methods section. Each irrelevant shRNA was transfected independently with the luciferase target to monitor non-speciﬁc eﬀects. Each Luc co-transfection experiment is normalized to its corresponding target-irrelevant control and to the Renilla luciferase transfection control. However, for convenience only one bar is shown to represent the target for each group and it is indicated by FLT+Irr (Irrelevant). Bluescript was used to adjust the total amount of DNA transfected in each sample. TAR indicates the co-transfection of a U6 construct expressing a mutated HIV-1 TAR structure (see text). (b) Non-saturating amounts of shRNAs can still compete for incorporation into RISC. A titration of U6 shLuc (Luc) was used to establish a concentration in the linear range for activity (left side of the chart). Five nanogram of U6sh Luc were then selected to be co-transfected with increasing amounts of a shRNA competitor (shSII) (right side of the chart). The numbers 1–25 indicate the amount in nanograms used in the reactions. optimal target down-regulation (Figure 2b). Based on these results, we co-tranfected 5 ng of shLuc with increasing amounts of the shSII competitor. The results show that even 1 ng of added competitor is able to reduce the activity of shLuc (Luc-5 + shSII-1, Figure 2b). SiRNAs compete for incorporation into RISC The observation that the shRNA competition increases with the total amount of transfected shRNA could be explained by the increasing saturation of Exportin 5, although the diﬀerent relative strengths of each shRNA to compete against the shLuc when used in equal amounts suggest that they are more likely due to a preferential incorporation into RISC. Moreover, the shRNA can also compete when used in non-saturating conditions (Figure 2b). However, to bypass Exportin 5 we repeated the co-transfection experiment using synthetic siRNAs (siH3, siH6 and siH1; Figure 3a). The luciferase target was transfected alone or with each synthetic siRNA (Figure 3a). The results of this experi- ment show that the synthetic siRNAs were able to aﬀect the down-regulation of the luciferase target when co-transfected with the Luc construct. Similar to what was shown with the shRNAs, the siRNAs competed to diﬀering degrees with the Luc shRNA. Since synthetic siRNAs do not need to be transported from the nucleus to the cytoplasm for their function and their activity is not aﬀected by Exportin-5 (20), the observed competition must be at the level of incorporation into RISC. Although a recent report shows that synthetic siRNAs can potentially shuttle from the cytoplasm back into the nucleus using Exportin-5 (29), this should not be a major competition pathway for synthetic siRNAs. Therefore, these results show that sh and siRNAs compete at the level of incorporation into RISC but may also compete for the Exportin 5 pathway to some extent. Sh and siRNAs compete for Exportin 5 To characterize any contribution made by the saturation of Exportin-5 on the competition detected among inter- fering RNAs, we included a plasmid-expressing Exportin- 5 in the co-transfection experiments. It has previously been shown that co-transfecting Exportin-5 relieves the saturation of transport and improves shRNA cleavage of its target (19,24). As expected, our results show that the overall activity of shLuc is improved, but competition among the diﬀerent sequences still persists (Figure 3b). It should be noted that for siRNAs, the eﬀect could be indirect, and based only on more eﬃcient export of shLuc. Micro RNAs do not compete for RNAi components Our results show that when diﬀerent exogenously supplied interfering RNAs are introduced into cells, competition for export and incorporation into RISC can decrease or completely block the activity of some RNAi triggers. These ﬁndings suggest that transfected siRNAs and shRNAs can also compete with endogenous microRNAs. To test this hypothesis, we inserted the siRNA directed against the site II HIV-rev-1 sequence (SII) into the backbone of the mir-30 microRNA. We made two diﬀerent versions of this microRNA, one containing the entire wild-type backbone (Mir-B, and the other with some additional mutations designed to