Figure 3. (a) Synthetic siRNAs can reduce the ability of a shRNA to down-regulate its target. A 10 nM concentration of each synthetic siRNA (siH3, siH6 and siH1) was co-transfected with the ﬁreﬂy luciferase target (FLT) independently or together with the anti-ﬁreﬂy shRNA (Luc). The ﬁreﬂy luciferase units were normalized against Renilla luciferase, which was used as transfection control. (b) Over- expression of Exportin-5 decreases shRNA competition but does not eliminate competition among interfering RNAs. Twenty nanomolar of each synthetic siRNA (siH3, siH6, siH1) or 50 ng of each shRNA construct (shL, shSII, shSI) were co-transfected with the luciferase target and the Luc shRNA with or without 100 ng of the Exportin-5- expressing plasmid (Exp-5) as indicated in the ﬁgure (+Exp-5, -Exp-5). Fireﬂy luciferase units were calculated as previously described. increase the stability of the microRNA structure (Mir- CG). Both microRNAs are processed (Figure 4c and data not shown) and are eﬀective in down-regulating a RLT which contains the corresponding complementary target sequence within its 30 UTR (Mir-B, Mir-CG; Figure 4a). When these microRNAs were co-transfected with an irrelevant shRNA-expressing construct (shSI), their inhibitory activity was dramatically reduced (Mir- B+shSI, Mir-CG+shSI; Figure 4a). Conversely, the microRNA constructs did not exert signiﬁcant inhibition on the activity of shRNAs as shown by co-transfecting Mir-B and Mir-CG with a U6 shRNA targeting the ﬁreﬂy luciferase gene (compare Luc to Luc+Mir-B, Luc+Mir-CG; Figure 4b). Importantly, the guide Nucleic Acids Research, 2007, Vol. 35, No. 15 5159 sequence expressed by the microRNA becomes a good competitor when it is expressed in the form of a shRNA (shSII, Figure 1a). Finally, when Mir-B and Mir-CG were co-transfected with an irrelevant microRNA-expressing construct (Mir- Irr), there was no signiﬁcant change in their ability to down-regulate their target (Mir-B+Mir-Irr, Mir- CG+Mir-Irr; Figure 4a), indicating that perhaps the safest method for down-regulation of cellular targets is to express the siRNA of choice within a microRNA backbone. To test if the diﬀerences among sh and microRNAs in their ability to compete resides primarily with their levels of expression, we repeated the competition experiment using concentrations that would generate comparable amounts of mature sequences as determined by Northern blot analysis (Figure 4c). A 4-fold excess of mirCG over shSII was used for the northern blot. Based on the diﬀerences in the processed SII sequence, which is expressed from both constructs, a 100-fold excess of mirCG over shSII was used in a competition assay (Figure 4c). The data show that a siRNA sequence is not an eﬀective competitor when expressed from a microRNA and yet it is very proﬁcient in down-regulating its target even when transfected at lower concentrations (Figure 4a). Si and shRNAs can compete with endogenous microRNAs To conﬁrm that si and shRNAs are able to interfere with the microRNA regulatory activities in an intracellular environment, we constructed a stable clonal cell line with an integrated EGFP gene that includes the target site for mir21 at its 30 end (HCT116-GFPmiR21). In this cell line, the EGFP gene is silenced by the endogenous expression of the mir21 microRNA. Any inhibition with the processing, transport and/or incorporation of mir21 would cause a reactivation of EGFP expression. It has previously been shown that 20O-Me-oligonucleotides (Antagomirs) can block microRNA activity when designed to bind the microRNAs’ guide sequences (30,31). As expected, transfection of an Antagomir fully complementary to the mir21 guide sequence in the HCT116-GFPmiR21 cells reactivated EGFP ﬂuorescence by blocking the activity of the endogenous mir21 (Mir-21– 20 O-Me, Figure 4d), while an irrelevant Antagomir used as control did not have any eﬀect on the level of ﬂuorescence (IRR-20O-Me, Figure 4d). Both the non- speciﬁc sh and siRNAs were able to increase EGFP ﬂuorescence, demonstrating that these constructs can interfere with the endogenous microRNA pathway (shSII, siH1, Figure 4d). However, when the same siRNA guide sequence was expressed from a microRNA backbone, it did not aﬀect the mir21 activity, as shown by the lack of EGFP ﬂuorescence (Mir-CG, Figure 4d). Two additional microRNA constructs were transfected in HCT116-GFPmiR21 with the same experimental outcome (data not shown). To conﬁrm that competition can alter the eﬃcacy of shRNAs when used in a combinatorial fashion, we tested