5252 Nucleic Acids Research, 2010, Vol. 38, No. 15 for the unmodiﬁed duplex) after modiﬁcation of either strand with four LNA nucleotides (PR-11 LN, PR-11 NL) (Figure 7B). The potency was reduced to the same extent whether the guide or passenger strand was modiﬁed. For activation by PR-11, all of the thermo- dynamically biased LNA analogs maintained signiﬁcant activity (Figure 7C). Modiﬁcation of either end of the guide strand (PR-11 NL3, PR-11 NL5) gave activity in- distinguishable from that of the native PR-11. Passenger strand modiﬁcation (PR-11 L3N, PR-11 L5N) gave a slight reduction in activation, and modiﬁcation of both strands (PR-11 L3L5, PR-11 L5L3) led to a further reduc- tion in activation. Nonetheless, even these duplexes modiﬁed in both strands gave >3-fold activation at the RNA level and clear activation at the protein level. Together with results of 20F and 20-OMe substitution, these data suggest that agRNA-mediated gene activation is broadly tolerant of chemical modiﬁcation. Eﬀect of combining modiﬁcations on gene silencing Many examples exist of potent and stable siRNAs containing multiple types of chemical modiﬁcations (38,54–56). In some cases, combination of multiple modi- ﬁcations led to potency and stability unattainable by the individual modiﬁcations alone (54,56). We set out to explore whether combination agRNA duplexes (Figure 8A) would yield better gene inhibition or activation in their respective cell lines. Even if existing potency is only maintained, the combination of diﬀerent chemistries could aﬀord better stability and other desir- able drug properties. Our criteria for choosing combinations was to select modiﬁed strands from PR-9 and PR-11 analogs showing the highest activity and recombine them in new ways. For PR-9 analogs, this led to the identiﬁcation of several potent new duplexes (Figure 8B). Duplexes including PR-9 L5L5, PR-9 JL, PR-9 JZ and PR-9 LZ retained sub- stantial activity. Duplexes PR-9 LL5 and PR-9 PpJ lost most activity. The eﬀects appear complex and cannot simply be characterized by considering additive eﬀects from the individual modiﬁcations. Nonetheless, it appears that terminal 20F-RNA modiﬁcation (Z) on the guide strand and LNA modiﬁcation at the 50-end of the passenger strand (L5) are generally well-tolerated, whereas 20OMe modiﬁcation of all pyridimines (P) on the passen- ger strand tends to disrupt the activity. It is noteworthy that two of the most active combin- ation duplexes also had Tm values that were too high for us to measure (PR-9 L5L5 and PR-9 JL). It is sometimes assumed that high Tm values prevent the strand separation required for siRNA/agRNA activity, but our ﬁndings conﬁrm that the relationship between Tm and activity is more complex. This result is consistent with our previous observation that the limited number of agRNA duplexes Figure 8. PR gene expression can be silenced by agRNAs containing combinations of modiﬁcations. (A) Duplex sequences: passenger strands are listed on top in all cases; for agRNA PR-9 this is the antisense strand. LNA sequences contain 5-methylcytosine instead of cytosine. Modiﬁcation codes: dna, RNA, 20F-RNA, 20Ome-rna, lna; p = phosphate. (B) PR-9 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.