5254 Nucleic Acids Research, 2010, Vol. 38, No. 15 Figure 10. Single strands of native or modiﬁed agRNAs are inactive. (A) Sequences: passenger strands are given 50-30, guide strands are given 30-50 as shown. For these agRNAs the passenger strand is the antisense strand. Modiﬁcation codes: dna, RNA, 20F-RNA, 20Ome-rna, lna; p = phosphate. (B) PR-9 duplex and native or modiﬁed single-strands. (C) PR-11 duplex and native or modiﬁed single-strands. All data are normalized to the guide strand of mismatch control MM4. RNA levels are the average (±SD) from at least two independent transfections. Protein levels were also conﬁrmed by at least two independent transfections, from which a typical western blot is shown. comparable potency to PR-9 itself (e.g. PR-9 NL). Others, while still active, showed reduced potency (e.g. PR-9 NZ). Modiﬁed duplexes aﬀect recruitment of RNA polymerase II We have previously used ChIP of RNA polymerase II (RNAPII) [see the supporting information of ref. (28)] and nuclear run-on assays (25) to show that both silencing and activating agRNAs aﬀect PR transcription. To inves- tigate whether chemically modiﬁed agRNAs operate through the same mechanism as their native counterparts, we carried out RNAPII ChIP using two active modiﬁed duplexes (Figure 12). In T47D cells, treatment with either PR-9 or its modiﬁed analog PR-9 PN led to reduced occupancy of RNAPII at the PR promoter relative to cells treated with the luciferase control duplex siGL2 (Figure 12A). In MCF7 cells, treatment with either PR-11 or the modiﬁed analog PR-11 pYN increased levels of RNAPII at the PR promoter (Figure 12B). The ﬁnding that native and modiﬁed duplexes behave similarly supports the hypothesis that they act through similar mechanisms. Native and modiﬁed duplexes have the same eﬀect on antisense transcript levels We have previously observed that both activating and in- hibitory agRNAs bind to an antisense transcript that overlaps the PR promoter (28). This transcript originates within PR mRNA and extends 70 000-bp upstream. It is spliced, and the spliced product contains target sequences for all the agRNAs used in this study. When biotinylated agRNAs are transfected into cells they bind the antisense transcript. RNA immunoprecipitation shows that both activating and silencing agRNAs recruit AGO to the anti- sense transcript. The antisense transcript, therefore,