The ﬁrst letter describes the chemical modiﬁcation of the passenger strand, while the second letter describes modiﬁ- cation of the guide strand. A lowercase ‘p’ indicates that the 50-end of the strand is phosphorylated. For example, a duplex labeled ‘NpF’ has a native passenger strand and a guide strand that is phosphorylated and fully modiﬁed with 20-ﬂuoro (20F-RNA) nucleotides. Passenger strands are listed above guide strands throughout. Eﬀects of 20F-RNA modiﬁcation on gene silencing The structure of 20F-RNA (Figure 1) is similar to that of RNA (31) but confers resistance to endonucleases (32,33). For siRNAs, partial modiﬁcation with 20-F RNA is tolerated throughout either strand of the duplex (34–36) and there have been reports of functional siRNAs that are extensively or completely modiﬁed with 20F-RNA (33,37,38). 20F-RNA has been shown to reduce the immunostimulatory activity of siRNAs (39,40). We tested three types of 20F-RNA-modiﬁed strands: fully modiﬁed (F), modiﬁed at all of the pyrimidine units (Y), and modiﬁed at positions near the termini (Z) (Table 1 and Figure 2A). Relative to unmodiﬁed duplex PR-9, we observed reduced potency when either strand was replaced with a fully-ﬂuorinated (PR-9 NpF, PR-9 pFN) or pyrimidine-ﬂuorinated strand (PR-9 NpY, PR-9 pYN) (Figure 2B). siRNA PR2526, by contrast, retained most silencing activity after total or partial modiﬁcation of its passenger strand (PR2526 pFN, PR2526 pYN) but activity was lost after modiﬁcation of the guide strand (PR2526 NpF, PR2526 NpY) (Figure 2B). Duplexes based on either PR-9 or PR2526 containing heavy modiﬁcation in both strands were largely inactive (Supplementary Figure S1). Given the loss of activity after aggressive modiﬁcation of PR-9, we tested more limited terminal substitutions. A Table 1. Descriptive strand names used throughout this work Abbreviation Strand chemistry N F Y Z P J L L3 L5 p Native RNA strand 20F-RNA, fully modiﬁed 20F-RNA, all pyrimidines 20F-RNA, contains three units near termini 20-OMe-RNA, all pyrimidines 20-OMe-RNA, two units at the 50-end LNA, contains four units distributed evenly LNA, contains two units toward the 30-end LNA, contains two units toward the 50-end A lower case ‘p’ preceding any strand abbreviation indicates a 50-phosphate Figure 1. Chemical modiﬁcations used in this study. Nucleic Acids Research, 2010, Vol. 38, No. 15 5245 PR-9 analog containing minimal modiﬁcation of the guide strand was eﬀective when paired with an unmodiﬁed passenger strand (PR-9 NZ) but not when the passenger strand was heavily modiﬁed (PR-9 YZ and PR-9 FZ) (Figure 2C). Analogs of PR2526 containing a minimally modiﬁed guide strand (PR2526 NZ, PR2526 YZ, PR2526 FZ) were active regardless of the extent of modi- ﬁcation of the passenger strand (Figure 2C). We also examined the eﬀect of 20F-RNA modiﬁcation on the activity of silencing agRNA PR-26 in an eﬀort to determine how modiﬁcation eﬀects might vary depending on the sequence of the agRNA. When the pyrimidines of either or both strands of PR-26 were replaced with 20F-RNA (PR-26 YN, PR-26 NY, PR-26 YY), most silencing activity was retained (Figure 2D). Taken together, data from PR-9 and PR-26 demonstrate that 20F-RNA substitutions are compatible with the mechan- ism for agRNA activity but that the activity of individual patterns of modiﬁcation can vary between duplexes. Eﬀect of 20F-RNA modiﬁcation on gene activation We introduced 20F-RNA into activating agRNA PR-11 (Figure 3A) and observed that the modiﬁcation was broadly tolerated. Modiﬁcation of the pyrimidines of the passenger strand (PR-11 pYN), total modiﬁcation of the passenger strand (PR-11 pFN), or heavy modiﬁcation of the guide strand (PR-11 NpF and PR11 NpY) all led to gene activation, albeit at levels below those of the unmodi- ﬁed PR-11 duplex (Figure 3B). Combining the unmodiﬁed or heavily modiﬁed passenger strands with a minimally modiﬁed guide strand showed a loss of potency as the degree of modiﬁcation increased (Figure 3C). Nevertheless, all of these duplexes retained signiﬁcant activity (2-fold to 6-fold at the RNA level). PR-11 analogs with both strands heavily modiﬁed were inactive (Supplementary Figure S2). To assess the generality of 20F-RNA modiﬁcation, we also tested a second activating agRNA, PR-22. For PR-22, 20F-RNA was well-tolerated in the passenger strand and duplexes PR-22 FN and PR-22 YN were able to activate gene expression at both RNA and protein levels without loss of potency (Figure 3D). The guide strand of PR-22 was more sensitive to 20F-RNA modiﬁcation, with lower levels of gene activation by agRNAs PR-22 NY and PR-22 YY. Taken together with results from PR-11, these results show that 20F-RNA substitutions can be well tolerated by activating agRNAs and that chemical modiﬁcations on the passenger strand tend to be better tolerated.