thermodynamically biased LNA analogs, to explore whether their activities would diﬀer (Figure 6C). Strand L3 contains LNA at the second and sixth positions from its 30-end, while strand L5 contains LNA at the second and sixth positions from its 50-end. Modiﬁcation of the 30-end of the guide strand (PR-9 NL3), reduced activity of PR-9 (Figure 6C). All other RNA duplexes containing two LNA substitutions on one strand (PR-9 NL5, PR-9 L3N, PR-9 L5N) were active regardless of which strand was modiﬁed or the location of the modiﬁcation. Some activity was lost when both strands were modiﬁed with LNA (PR-9 L3L5, PR-9 L5L3). Variants of PR2526 containing LNA bases yield results that were analogous to LNA-substituted variants of PR-9 (Figure 6B and D). These results demonstrate that LNA modiﬁcations are compatible with gene silencing by agRNAs, but show no Nucleic Acids Research, 2010, Vol. 38, No. 15 5251 evidence for biased strand loading in the context of the PR-9 or PR2526 sequences. The reduction in activity upon modiﬁcation of the 30-end of the guide strand (‘NL3’) is consistent with previous observations that LNA modiﬁca- tion of the sixth nucleotide from the 30-end of this strand leads to reduced potency (51). Results were similar for each pattern of modiﬁcation regardless of whether agRNA PR-9 or siRNA PR2526 was the parent duplex, emphasizing that the response of agRNAs and siRNAs to chemical modiﬁcation can be similar. Eﬀect of LNA on gene activation We tested LNA-modiﬁed analogs of PR-11 for gene acti- vation in MCF7 cells (Figure 7A). For activating duplex PR-11, we observed activation (2- to 3-fold versus 6-fold Figure 7. PR gene expression can be activated by agRNAs containing LNA. (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, lna. (B) PR-11 analogs containing four evenly distributed LNA units. (C) PR-11 analogs containing LNA at strand ends. 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.