Figure 1.
Anti-miRs specifically associate with the Argonaute in vivo and in vitro, only in the context of the cognate target miRNA.
a) Northern analysis of anti-miR-122 in input lysate and in immunopurified Argonaute-containing complexes (IP) from liver tissue of animals dosed subcutaneously, three days prior, with 1, 3 or 10 mg/kg of the compound. Representative experiment shown from 2 independent experiments. b) Western analysis of the levels of Argonaute associated with biotinylated compounds in liver lysates from wild-type and miR-21-deficient animals. 10,3, or 1 pmoles of the 5′-biotinylated compounds were incubated with liver lysates, purified with streptavidin beads and the associated Argonaute proteins were visualized with an anti-Argonaute antibody. Representative experiment shown from three independent experiments. c) Quantification of Argonaute levels associated with the compounds from panel (b). d) Luminescence levels of a miR-21 probe bound by immunopurified miRNA:Argonaute complexes from wild-type or miR-21-lacking liver extracts (miR-21-/-), in the presence of increasing amounts of anti-miR-21. A miR-122 probe in the presence of increasing amounts of anti-miR-122 was used as a positive control for the assay. Representative experiments shown from 3 independent experiments (n = 3).
Figure 2.
The seed region is an important determinant for anti-miRs to associate with the miRNA:Argonaute complexes in vitro and inhibit the target miRNA in vivo.
a) Western analysis of the levels of Argonaute from liver lysates that associate with an anti-miR-122 compound with perfect seed matching (match) or with 3 mismatches in the seed region (mismatch). 10,3, or 1 pmoles of the 5′-biotinylated compounds were incubated with liver lysates, purified with streptavidin beads and the associated Argonaute proteins were visualized with an anti-Argonaute antibody. Representative experiment of 3 independent experiments. b) Luminescence levels of a miR-122 probe bound by Argonaute in the presence of an anti-miR-122 compound with perfect seed matching and its seed-mismatched counterpart (3 mismatches) or an unrelated negative control sequence. Match and mismatch sequences are as in (a). Representative experiment shown from 3 independent experiments (n = 3). c) mRNA levels of the miR-122-regulated Aldoa transcript in livers of mice treated with PBS or anti-miR-122 compounds with mismatches as shown in the table. Mice were treated subcutaneously for ten days with 25 mg/kg of the indicated compound. Results are shown as percentage of change over the PBS-treated animals ± s.d. Representative experiment from 2 independent experiments (n = 5). d) Aldoa mRNA levels in animals treated with anti-miR-122 compounds targeting the seed plus 6 complementary or random nucleotides. PBS controls or anti-miR solutions were administered subcutaneously at 25 mg/kg. Representative experiment from 2 independent experiments (n = 5).
Figure 3.
Subtle changes in the chemical modification pattern can affect anti-miR association with Argonaute and anti-miR activity in vitro and in vivo.
a) Western analysis of the levels of Argonaute associated with anti-miR-21-A or anti-miR-21-B compounds, in liver lysates. These two compounds share the same sequence and chemistry, a phosphorothioate backbone with DNA and constrained Ethyl (red) bases. 10,3, or 1 pmoles of the 5′-biotinylated compounds were incubated with liver lysates, purified with streptavidin beads and the associated Argonaute proteins were visualized with an anti-Argonaute antibody. Samples from the same gel. Representative experiment from 3 independent experiments. b) Luminescence levels of a miR-21 probe bound by Argonaute in a competition binding assay with increasing amounts of anti-miR-21-A, anti-miR-21-B, unlabeled probe or an unrelated sequence as control. miRNA:Argonaute complexes were purified from HeLa cells. Representative experiment from 3 independent experiments (n = 3). c) mRNA levels of the miR-21-regulated ANKRD46 gene in U87 cells transfected with increasing concentrations of anti-miR-21-A or anti-miR-21-B compounds. Results are shown as fold change over mock transfected ± s.e.m. Representative experiment from 2 independent experiments (n = 5). d) mRNA levels of the miR-21-regulated Rnf167 transcript in livers of mice treated with PBS or the anti-miR-21 compounds A or B. Mice were injected subcutaneously once with 50 or 100 mg/kg of anti-miR-21-A or B, RNA was extracted from liver samples three days post-injection, and analyzed for Rnf167 mRNA levels by qPCR. Results are shown as fold change over the PBS-treated group of animals ± s.d. Representative experiment from 2 independent experiments (n = 5).
Figure 4.
Anti-miR treatment decreases the levels of Argonaute-bound mRNA targets, increasing their stability and abundance in vivo.
a and b) Cumulative-distribution fraction plots (CDF) depicting the mRNA fold change in total RNA (a) and in the Argonaute immunopurified (Ago) fraction (b) from liver lysates of animals dosed with with 10 1 mg/kg of anti-miR-122 as compared to PBS-treated controls as determined by microarray analysis. c and d) Total and Argonaute IP (Ago) RNA was assayed for the miR-122 targets (c) Aldoa and (d) Cd320. Mice were treated subcutaneously with 1, 3 or 10 mg/kg of an anti-miR-122 compound or PBS and livers were collected three days later. Results are displayed as fold-change±s.e.m.. Representative experiment from 2 independent experiments shown (n = 3).
Figure 5.
Anti-miRs do not affect the levels of total mature or Argonaute-bound miRNA in vivo.
Total liver RNA isolated from animals 7 days post-subcutaneous administration of anti-miR-122 (10 mg/kg). miRNA levels in total RNA sample, profiled on the Nanostring platform, before (a) and after (b) the glyoxal treatment to separate the miR/anti-miR-122 duplex which interferes with miR-122 detection. c and d) Global miRNA levels, post-glyoxal treatment and profiled on the Nanostring platform, in anti-miR-122-treated animals compared to PBS-treated controls from total RNA (c) or immunopurified Argonaute fractions (Ago) (d). Representative experiments shown from 2 independent experiments (n = 3).
Figure 6.
Model of the anti-miR mechanism of action.
miRNAs, loaded onto Argonaute, guide the miRISC complex to target mRNA transcripts. The anti-miR specifically associates with Argonaute, in the context of the cognate target miRNA and now the miRNA:Argonaute complex can no longer bind and regulate target mRNAs. The mRNA targets are stabilized and can now be translated.