Nucleic Acids Research, 2010, Vol. 38, No. 15 5255 Figure 11. Sample dose response experiments for both (A) silencing and (B) activation. More dose response experiments for PR-9 analogs are shown in Supplementary Figures S3 and S4. Table 2. IC50 values for PR-9 and modiﬁed analogs in T47D cellsa Silencing agRNA PR9 PR9 NZ PR9 JpJ PR9 PN PR9 NL PR9 L5N Type of chemistry native 20F 20OMe 20OMe LNA LNA IC50 (RNA) (nM) IC50 (protein) (nM) 5.6 18.6 9.4 12.9 6.8 13.3 12.2 19.6 10.2 12.6 13.2 7.4 aPlots used to derive these data are shown in Supplementary Figures S3 and S4. Figure 12. ChIP of RNA Polymerase II (RNAPII) shows that both native and modiﬁed agRNAs operate at the transcriptional level. (A) Treatment of T47D cells with PR-9 or PR-9 PN leads to reduced oc- cupancy of RNAPII at the PR promoter relative to cells treated with control duplex siGL2. (B) Treatment of MCF7 cells with PR-11 or PR-11 pYN leads to increased occupancy of RNAPII at the PR promoter relative to cells treated with control duplex siGL2. appears to play a central role in the mechanism of agRNAs. We explored the eﬀect of chemically modiﬁed agRNAs on levels of the promoter-associated antisense transcript. We previously observed that treatment of T47D cells with silencing agRNA PR-9 led to a reduction of antisense transcript levels, while treatment of MCF7 cells with activating agRNA PR-11 led to no signiﬁcant change in antisense transcript levels (28). We repeated these experi- ments using both the native duplexes and several of the most active modiﬁed analogs. We monitored expression of PR mRNA and the antisense transcripts that span the agRNA target region using qPCR. Native and modiﬁed duplexes gave comparable results. PR-9 and modiﬁed PR analogs reduced expression of both PR mRNA and the promoter-derived antisense transcript similarly (Figure 13A). Conversely, PR-11 and modiﬁed analogs increased expression of PR mRNA, but caused no signiﬁcant change in antisense transcript levels (Figure 13B). The ﬁnding that native and modiﬁed duplexes have the same eﬀect on levels of the antisense transcript supports the conclusion that they act through similar mechanisms. Inactive modiﬁed duplexes do not compete for target sites It is interesting to speculate as to why some patterns of modiﬁcation cause reduced activity. Inactive agRNAs could be failing at any of a number of steps: (i) loading into the RNA-induced silencing complex (RISC), (ii) cleavage or dissociation of the passenger strand, (iii) import into the nucleus, (iv) binding of the RNA target, or (v) execution of the eﬀect (activation or silencing). We previously observed that inactive native duplexes such as