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. 2006 Jan;12(1):163-76.
doi: 10.1261/rna.2150806. Epub 2005 Nov 21.

Small interfering RNAs containing full 2'-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

Bryan A Kraynack  1 , Brenda F Baker
Affiliations

Small interfering RNAs containing full 2'-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

Bryan A Kraynack et al. RNA. 2006 Jan.

Abstract

RNA interference (RNAi) is a process by which short interfering RNAs (siRNAs) direct the degradation of complementary single-strand RNAs. In this study, we investigated the effects of full-strand phosphorothioate (PS) backbone and 2'-O-methyl (2'-OMe) sugar modifications on RNAi-mediated silencing. In contrast to previous reports, we have identified active siRNA duplexes containing full 2'-OMe-modified sense strands that display comparable activity to the unmodified analog of similar sequence. The structure of these modified siRNAs is the predominant determinant of their activity, with sequence and backbone composition being secondary. We further show, by using biotin-tagged siRNAs and affinity-tagged hAgo2/eIF2C2, that activity of siRNA duplexes containing full 2'-OMe substitutions in the sense strand is mediated by the RNA-induced silencing complex (RISC) and that strand-specific loading (or binding) to hAgo2 may be modulated through selective incorporation of these modifications.

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Figures

FIGURE 1.
FIGURE 1.
Activity of 19-bp 3′-dTdT siRNA duplexes with full 2′-O-methyl sense strands is sequence-dependent. Dose response analyses of 19-bp 3′-dTdT siRNAs targeting Site I (A), Site II (B), Site III (C), and Site IV (D) of the PTEN mRNA in HeLa cells. The sequence and structure of siRNAs are shown to the left of each graph and are unmodified oligoribonucleotides unless indicated otherwise. (*) A phosphorothioate (PS) modification, (underline) 2′-OMe-modified nucleotides, (dT) 2′-deoxythymidine. HeLa cells were treated with siRNAs at 0.6, 3, 15, and 75 nM, as detailed in Materials and Methods. PTEN mRNA levels were determined relative to c-raf mRNA levels by real-time RT-PCR (TaqMan). Results shown represent the percentage of untreated control expression (UTC). Each bar represents the mean of three biological replicates (±standard deviation).
FIGURE 2.
FIGURE 2.
Activity of siRNAs with full 2′-O-methyl sense strands is dependent on construct design. Dose response analysis of two types of siRNA constructs (19-bp 3′-dTdT vs. blunt-end 20 bp) targeting each of the four PTEN mRNA target sites in HeLa cells. The construct structure and sequence are shown to the left of each graph and are unmodified oligoribonucleotides unless indicated otherwise. (Underline) 2′-OMe-modified nucleotides, (dT) 2′-deoxythymidine residues. HeLa cells were dosed at 0.6–75 nM with siRNA, as described in Materials and Methods. The results shown represent the percentage of untreated control expression (UTC). Each bar represents the mean of three biological replicates (±standard deviation).
FIGURE 3.
FIGURE 3.
Blunt-end 20-bp siRNAs with full 2′-O-methyl sense strands and full PS backbone modifications mediate target mRNA reduction. Dose response analyses of modified blunt-end 20-bp siRNAs comprised of full 2′-OMe-modified sense strands to Site I (A), full 2′-OMe-modified sense strands to Site III (B), and full 2′-OMe-modified anti-sense strands to Site I (C). The siRNA constructs are shown to the left of each graph and are unmodified oligoribonucleotides unless indicated otherwise. (*) A PS modification, (underline) 2′-OMe-modified nucleotides. The results shown represent the percentage of untreated control expression (UTC). Each bar represents the mean of three biological replicates (±standard deviation).
FIGURE 4.
FIGURE 4.
Modified siRNA activity is attenuated by knock-down of hAgo2. (A) Validation of ASO knock-down assay in HeLa cells. siRNA (I_1) was transfected into HeLa cells at a dose of 0.6–75 nM post-treatment with either the control ASO (ISIS 129686) or the ASO specific for hAgo2 (ISIS 136764). (B) Effect of hAgo2 knock-down on activity of siRNAs containing a full 2′-OMe-modified sense strand. Modified and unmodified siRNAs targeting PTEN Site III were transfected (75 nM) into HeLa cells previously treated with ISIS 129686 or ISIS 136764. PTEN and hAgo2 mRNA levels were determined relative to the cRAF mRNA level by real-time RT-PCR (TaqMan). The results shown represent the percentage of untreated control expression (UTC). Each bar represents the mean of three biological replicates (±standard deviation). The control ASO (ISIS 129686) is a random sequence that has no significant homology to the human genome.
FIGURE 5.
FIGURE 5.
Modified siRNAs interact with overexpressed hAgo2. (A) Characterization of pE2-N_HA. Total HeLa lysates prepared from untreated cells (lane 1) and cells expressing HA-hAgo2 (lane 2) were separated by SDS-PAGE and immunoblotted with anti-HA antibodies. The migration of the molecular mass standards is shown on the left. (B) Western blot analysis of biotin-siRNA samples isolated from HeLa cells expressing HA-hAgo2. Biotin-tagged strands were isolated from total cell lysates, as described in Materials and Methods. Isolates were then subjected to SDS-PAGE and Western blotted with anti-HA antibody. (C) Structure and sequence of siRNA constructs included in analysis. (*) A PS modification, (underline) 2′-OMe-modified nucleotides, (Bi) biotin residue.
FIGURE 6.
FIGURE 6.
Reduced association of full 2′-OMe anti-sense strands with overexpressed hAgo2. (A) Western blot analysis of biotin-siRNA isolated from HeLa cells expressing HA-hAgo2. Samples were subjected to SDS-PAGE and Western blotted with anti-HA antibody, as described in Materials and Methods. (B) Structure and sequence of siRNA constructs included in analysis. (*) A PS modification, (underline) 2′-OMe-modified nucleotides, (Bi) biotin residue.
FIGURE 7.
FIGURE 7.
Modified siRNAs enter into the hAgo2/RISC complex to mediate target RNA cleavage. (A) A 5′-[P32] end-labeled 40-mer substrate containing Site I was incubated with immunopurified RISC complexes, as described in Materials and Methods. The sequence of siRNA constructs and lane assignments are shown in B. The sequence of each RNA marker corresponds to the expected cleavage product of 23 nt (control site) or 17 nt (Site I). (*) A phosphorothioate modification, (underline) 2′-OMe-modified nucleotides. (C) Cleavage products were resolved on a 12% polyacrylamide 7 M urea gel. The 5′ cleavage products are indicated by arrows. Sequence identity was assigned according to the control digestions of the substrate by RNase T1, base (OH) hydrolysis, and synthetic RNA markers.
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