Targeted delivery of miR-146a mimic to RISC/Ago2 complexes in myeloid cells. (A) The design of C-miRNA146a mimic conjugate; underlined, phosphorothioated nucleotides; “o”, C3 units of the carbon linker; green, 2′-O-methyl-modified nucleotide. (B) Serum stability of miR146a (left) and C-miR146a (right). Oligonucleotides were incubated in 50% human serum at 37°C for the indicated times and then resolved on 7.5 M urea/15% polyacrylamide gel electrophoresis. Shown is a representative result from 1 of 3 independent experiments; the band intensities were quantified, and the estimated oligonucleotide half-lives (T_1/2) are indicated. (C) The intracellular uptake of C-miR146a^Cy3 compared with miR146a^Cy3 alone by primary human immune cells (monocytes, CD14⁺; myeloid dendritic cells [mDCs], CD1c⁺; plasmacytoid dendritic cells [pDCs], CD303⁺; B cells, CD19⁺; and T cells, CD3⁺), mouse RAW264.7 macrophages, human MDSL and HL-60 leukemia, human Raji lymphoma, and TAIL7 T-cell leukemia cells. Cells were incubated for 1 hour with 100 nM of C-miR146a^Cy3 or miR146a^Cy3 without any transfection reagents, and the uptake was measured by using flow cytometry. (D) The intracellular localization of C-miR146a^Cy3 or miR146a^Cy3 oligonucleotides (100 nM/red) as visualized by using confocal microscopy in RAW264.7 cells after 1 hour of incubation. Hoechst33342 (blue) was used for nuclear counterstain. (E) The successful loading of miR146a into RISC/Ago2 by C-miRNA conjugate. RAW264.7 or splenocytes isolated from miR-146a^−/− mice were incubated for 1 hour with 1 μM of C-miR146a or miR146a alone. The RNA-protein complexes were immunoprecipitated by using anti-Ago2 or control IgG antibodies, and the miR-146a levels were quantified by using qPCR. The equal protein loading was confirmed with western blotting. The data shown represent results from 3 independent experiments; shown are means ± SEM. ***P < .001 compared to untreated.