PR-8 (targeting –8 to +11) and PR-12 (–12 to +7) could compete for target sites with the active duplex PR-11 (23). Thus, treating MCF7 cells with inactive duplex PR-12 prevented gene activation by PR-11 in a subsequent trans- fection several days later. On the other hand, initial treat- ment with PR-11 followed later by PR-12 gave activation comparable to PR-11 alone (23). This experiment showed that the order of addition is crucial and the duplexes compete for the same target sites. Duplexes PR-8 and PR-12 failed at step (v) above—they were loaded into RISC and even bound the correct RNA target, but failed to execute an agRNA eﬀect. Therefore, since shifting the duplex–target interaction by 1–3 bp is suﬃ- cient to eliminate activity, the geometry of interaction at the promoter may be crucial for induction of gene activation. To test where inactive chemically modiﬁed agRNAs might be deﬁcient we carried out a similar experiment using two inactive modiﬁed duplexes each for silencing and activation. In T47D cells we used the inactive duplexes PR-9 YY and PR-9 pFN. In both cases, levels of PR expression were identical whether the inactive duplexes were transfected before or after the active duplex PR-9 (Figure 14A). Similarly, treatment of MCF7 cells with PR-11 gave gene activation regardless of whether the duplexes were transfected before or after inactive modiﬁed duplexes PR-11 PL and PR-11 LP (Figure 14B). These data suggest that the modiﬁed duplexes did not compete for target sites with native duplexes. Since the inactive modiﬁed duplexes are not binding the target site, they must be failing at one of the ﬁrst four steps in the process: RISC loading, passenger strand removal, nuclear import or target binding. Furthermore, some of the inactive modiﬁed duplexes contain native guide strands, thus it seems clear that they are failing at one of the ﬁrst two steps—RISC loading or passenger strand removal—since after this point they would be identical with native duplexes. Therefore in most cases, the challenges causing modiﬁed agRNAs to fail are the same as those faced by chemically modiﬁed siRNAs that are complementary to mRNA. agRNAs and chemical modiﬁcations Double stranded RNAs that are complementary to mRNA are currently being tested in clinical trials (1–4). Given the success of siRNAs in the laboratory and progress in the clinic, why would agRNAs enhance the potential for in vivo application of double-stranded RNA? One reason is that activating gene expression would open up a wider range of therapeutic targets and may provide new opportunities for development. For example, if more of a protein product is needed to treat a disease, upregulating expression using agRNAs might prove advantageous, especially if the protein’s site of action is intracellular and cannot be accessed by system- ically administered protein. Even in the case of gene silencing, where existing siRNA technology often gives superb results, it is possible that silencing at the transcrip- tional level may be advantageous for some targets. It is possible to envision that agRNAs may be more potent in Nucleic Acids Research, 2010, Vol. 38, No. 15 5257 some cases, produce longer lasting eﬀects, or produce diﬀerent modulation of isoform expression. Finally, understanding the eﬀect of modiﬁcations will also important for future studies of agRNA function in cultured cells. Potential applications for promoter- targeted duplex RNAs have been summarized in a recent review (66). Our data have several implications for the mechanism of chemically modiﬁed agRNAs: (i) As with any struc- ture–function analysis, the most important ﬁnding is that some designs yield active compounds whereas others do not; (ii) Two strands are necessary for eﬃcient action of agRNAs, we observe no silencing or activation by single strands at the highest concentrations used in this study; (iii) Activation and silencing show a regular, pre- dictable dose dependence; (iv) As with siRNAs, optimal modiﬁcations need to be determined empirically for each sequence; (v) As with siRNAs, both guide and passenger strands can be modiﬁed, but modiﬁcations are generally better tolerated on the passenger strand; (vi) IC50 values and maximal eﬃcacies of modiﬁed and unmodiﬁed agRNAs are similar, consistent with the expectation that they operate by a common mechanism; (vii) Both native and modiﬁed duplexes aﬀect recruitment of RNAPII, also consistent with a common mechanism; (viii) Addition of modiﬁed and unmodiﬁed agRNAs leads to almost identi- cal changes in antisense transcript levels, again consistent with a shared mechanism; and (ix) Inactive duplexes do not compete with active duplexes, suggesting that the inactive duplexes fail at a step prior to association with the antisense transcript. The ﬁnding that agRNAs containing 20F-RNA, 20OMe-RNA, or LNA retain the ability to silence or activate gene expression is signiﬁcant because it demon- strates that agRNAs tolerate the introduction of modiﬁ- cations known to be powerful tools for improving in vivo properties. For duplex RNAs that are complementary to mRNA, it is commonly observed that diﬀerent sequences tolerate diﬀerent chemical modiﬁcations or diﬀerent patterns of the same chemical modiﬁcation. The interplay of chemical modiﬁcations with each particular sequence is the ultimate determinant of duplex potency and duplexes with optimal properties must be determined empirically. No universal rules of siRNA modiﬁcation have been de- veloped because our understanding of the nature of this interplay is not yet suﬃcient. Our data on agRNAs suggest the same conclusion. We have tested two diﬀerent silencing agRNAs, PR-9 and PR-26, and two diﬀerent activating agRNAs, PR-11 and PR-22. While both tolerate a wide range of modiﬁcations, the relative potencies of analogous modiﬁed duplexes diﬀer. Like siRNAs, we conclude that empirical testing will be needed to identify agRNAs with the best balance of properties. While empirical testing may be necessary, we found that a wide range of modiﬁcations were compat- ible with gene silencing or activation by an agRNA-mediated mechanism and we predict that it will not be diﬃcult to develop active chemically modiﬁed agRNAs for genes of interest where a parent unmodiﬁed agRNA has already been identiﬁed.