5248 Nucleic Acids Research, 2010, Vol. 38, No. 15 Eﬀect of 20-O-methyl-RNA on gene silencing 20-O-Methyl-RNA (20OMe) nucleotides (Figure 1) improve the thermal and nuclease stability of siRNA duplexes (41,42). Inclusion of 20OMe-RNA units also reduces oﬀ-target eﬀects mediated either through the miRNA pathway (5) or the innate immune system (43– 45). We tested duplex RNAs containing two 20OMe ribonucleotides at the 50-terminus of each strand (J) or at all pyrimidine nucleotides (P) (Figure 4A). Duplexes PR-9 JJ, containing 20OMe nucleotides at both 50-termini, and PR-9 PN, containing 20OMe modiﬁcation at the pyrimidine nucleotides of the passenger strand, were both eﬀective gene silencing agents (Figure 4B). Duplexes containing pyrimidine modiﬁcations on the guide strand (PR-9 NP, PR-9 PP) were inactive, consistent with previous observations that 20OMe modiﬁcation is often better tolerated in the passenger strand (41). Variants of PR2526 (PR2526 PN, PR2526 NP and PR2526 PP) had activities similar to the PR-9 variants (Figure 4B). We also examined the eﬀects of 20OMe modiﬁcation of a second silencing agRNA, PR-26, and identiﬁed analogs that inhibited PR expression (Figure 4C). However, for PR-26, modiﬁcation of the guide strand in PR-26 NP did not prevent gene silencing. These data demonstrate that agRNA-mediated gene silencing can tolerate the introduction of 20OMe-RNA modiﬁcations. The inﬂuence of a speciﬁc pattern of modiﬁcation, however, depends on the identity of the parent sequence and can vary between agRNAs. Eﬀect of 20-O-methyl-RNA on gene activation We introduced 20-O-methyl modiﬁcations into two diﬀer- ent activating agRNAs, PR-11 and PR-22 (Figure 5A). Substitution of 20OMe units into PR-11 reduced activa- tion from 8-fold to 2–3-fold (Figure 5B). PR-11 PP with two heavily modiﬁed strands were the least activating. By contrast, activation by modiﬁed PR-22 duplexes was rela- tively stable at 4- to 6-fold regardless of modiﬁcation Figure 4. PR gene expression can be silenced by agRNAs containing 20OMe-RNA. (A) Duplex sequences: passenger strands are listed on top in all cases; for siRNA PR2526 this is the sense strand, but for agRNAs PR-9 and PR-26 this is the antisense strand. Modiﬁcation codes: dna, RNA, 20Ome-rna. (B) PR-9 analogs and controls containing 20OMe-RNA. (C) PR-26 analogs containing 20OMe-RNA. All data are normalized to mismatch control MM4. RNA levels are the average (±SD) from at least two independent transfections. *P < 0.05, **P < 0.01, ***P < 0.005. Protein levels were also conﬁrmed by at least two independent transfections, from which a typical western blot is shown.