Figure 5.

Motif-dependent activity of 2′OMe AMO is TLR7 specific. (A) mFOLD-predicted structure and free energy (ΔG, in kcal/mol) of miR-224–5p 2′OMe AMO used at 37°C (31). The bases of the consensus inhibitory motif are shown in red. Arrows point to mutations introduced in miR-224–5p AMOs to create the inhibitory motif in miR-224–5p mut2. BMMs from WT mice (B) and human PBMCs (C) pre-treated with 40 nM of the indicated 2′OMe AMO or RD, were stimulated overnight with 180 nM of B-406AS-1 (B and C) or β-Gal-656-REV (B), and TNF-α levels were measured in supernatants by ELISA. (B) The data are averaged from two (for β-Gal-656-REV) or three (for B-406AS-1) independent experiments, in biological triplicate. Percentages of cytokine production compared to the RD+B-406AS-1 (black) or β-Gal-656-REV condition (white) are given. (C) The data are averaged from three independent experiments in different blood donors, in biological triplicate. (D) HEK293 cells stably expressing TLR7 or TLR9 and an NF-κB-luciferase reporter, were transfected with 200 nM of the indicated AMO for 45 min prior to stimulation with R848 (200 ng/ml) (white) or polyI:C (20 μg/ml) (grey) for TLR7 cells, and ODN 2006 (400 nM) (black) for TLR9 cells. Firefly luciferase was assayed after overnight incubation as described in the Materials and Methods section. Luminescence reads normalized to unstimulated cells (NT) are shown as percentages of the RD+TLR ligand condition. The data are averaged from a minimum of three independent experiments in biological triplicate. (B–D) Ordinary one-way ANOVA with Dunnett's multiple comparison tests to the miR-224–5p+B-406AS-1 condition are shown. Unless otherwise indicated, differences were not significant. SEM is shown.