tested to date do not rigidly adhere to published guidelines for predicting the activity of duplex RNAs (23). We combined modiﬁed strands from active PR-11 analogs to give a new series of activating agRNAs (Figure 9A). All the combination duplexes based on PR-11 lost signiﬁcant activity with respect to the unmodi- ﬁed control. Duplexes containing LNA in the passenger strand and 20F-RNA at the termini of the guide strand (PR-11 LZ and PR-11 L5Z) were the best of this series, but only showed 2- to 3-fold activation at the RNA level (Figure 9B). No evidence that single-strands mediate agRNA activity There have been reports that single-stranded antisense RNA can lead to inhibition via the RNAi pathway (12,57,58). In addition, previous work from our lab (59–61) and others (62–65) has shown that single-stranded oligonucleotides including peptide nucleic acid (PNA) and LNA–DNA chimeras were able to bind chromosomal DNA and repress gene expression at the transcriptional level. To explore whether the single-stranded RNAs con- tained within agRNAs can mediate such eﬀects, we trans- fected T47D and MCF7 cells with various native and modiﬁed single strands from functional silencing or activating agRNA duplexes (Figure 10A). Transfections were carried out under the same conditions and at the same concentration (25 nM) as duplex RNAs. Results from both qPCR and western blots demonstrate that single-stranded agRNA, when transfected alone, had no Nucleic Acids Research, 2010, Vol. 38, No. 15 5253 eﬀect on silencing or activation of PR gene expression (Figure 10B and C). These data demonstrate that potent modulation of gene expression by anti-PR agRNAs requires the presence of intact duplex RNA regardless of chemical modiﬁcation. Our data indicate that gene silencing or activation by the agRNA in our study is not due (i) to slight excess of one strand, (ii) to degradation of one strand (prior to incorporation into AGO), or (iii) to single-strand-mediated oﬀ-target eﬀects. The requirement for RNA duplexes also provides further evidence that both native and chemically modiﬁed agRNAs make use of the RNAi machinery. Concentration dependence of modiﬁed duplexes To understand the eﬀect of chemical modiﬁcation on the potency of agRNAs we examined the dependence of gene silencing or activation on the concentration of selected RNAs. We observed that potencies of gene silencing by modiﬁed agRNAs PR-9 L5N and PR-9 NZ were similar to parent agRNA PR-9 (Figure 11A, see also Supplementary Figures S3 and S4). Likewise, potencies of gene activation by PR-11 pYN and PR-11 pFN were similar to gene activation by parent agRNA PR-11 (Figure 11B). Thus it is possible to achieve potent, dose-dependent gene modulation by both activating and inhibitory chemically modiﬁed agRNAs. For silencing agRNAs based on PR-9, we calculated IC50 values for the native duplex and several modiﬁed analogs (Table 2) (Supplementary Figures S3 and S4). At both the protein and RNA levels, some of the modiﬁed analogs were of Figure 9. PR gene expression can be activated by agRNAs containing combinations of modiﬁcations. (A) Duplex sequences: passenger strands are listed on top in all cases; for agRNA PR-11 this is the antisense strand. LNA sequences contain 5-methylcytosine instead of cytosine. Modiﬁcation codes: dna, RNA, 20F-RNA, 20Ome-rna, lna. (B) PR-11 analogs containing combinations of previously identiﬁed active strands. All data are normalized to 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.