Nucleic Acids Research, 2010, Vol. 38, No. 15 5249 Figure 5. PR gene expression can be activated by agRNAs containing 20OMe-RNA. (A) Duplex sequences: passenger strands are listed on top in all cases; for agRNAs PR-11 and PR-22 this is the antisense strand. Modiﬁcation codes: dna, RNA, 20Ome-rna. (B) PR-11 analogs containing 20OMe-RNA. (C) PR-22 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. (Figure 5C). These data suggest that, as observed above for gene silencing, the impact of 20OMe modiﬁcations on gene activation will vary depending on the parent sequence. Eﬀect of LNA on gene silencing Locked nucleic acid (LNA) (Figure 1) is a rigid nucleotide analog containing a constrained, bicyclic sugar. Introduction of locked nucleotides into DNA or RNA oligomers leads to substantially enhanced aﬃnities for complementary sequences (46–49) and enhanced resist- ance to digestion by nucleases (49–51). Several studies have also shown that the introduction of LNA nucleotides can improve the properties of siRNAs (9,35,51–53). LNA has been shown to improve loading of the desired strand (51), increase nuclease resistance (51) and lower immune stimulation (53). We tested a series of duplexes containing four LNA nucleotides evenly distributed across either the passenger or guide strand (Figure 6A). PR-9 LN and PR-9 NL both retained the ability to silence gene expression, albeit to a lesser extent than PR-9 (Figure 6B). Signiﬁcant silencing activity was maintained regardless of whether the sense or antisense strand was modiﬁed. The high binding aﬃnity of LNA has been used to direct loading of the appropriate strand into AGO and bias strand usage (51). We tested a series of