Design and cell-selective intracellular uptake of the CpG-STAT3 decoy oligonucleotide. (A) Predicted hairpin structure of the CpG-STAT3dODN consisting of CpG-D19 ODN linked to the double-stranded STAT3 decoy. Underlined are phosphothioation sites in the ODN backbone; o = single unit of the C3 carbon chain (CH₂)₃; asterisks (*) indicate fluorochrome conjugation sites (Cy3, 3′ end; FITC, in the linker). (B-D) Dose- and time-dependent internalization of CpG-STAT3dODN by target immune and leukemic cells without any transfection reagents. CpG(D19)-STAT3dODN or unconjugated STAT3dODN molecules were Cy3-labeled to detect their intracellular uptake by target cells using flow cytometry. (B) Human immune cells were incubated with indicated concentrations of CpG(D19)-STAT3dODN^Cy3 or STAT3dODN^Cy3 for 4 hours. The oligonucleotide uptake by CD14⁺ monocytes, CD1c⁺ (BDCA1⁺) mDCs, CD303⁺(BDCA2⁺) pDCs, CD19⁺ B cells, and CD3⁺ T cells was measured using flow cytometry. Similar results were obtained from 3 independent experiments. (C) Mouse RAW264.7 macrophages and CMM⁺ (Cbfb/MYH11/Mpl⁺) leukemia cells rapidly internalize CpG(D19)- and CpG(1668)-STAT3dODN conjugates. Cells were incubated with 250 nM Cy3-labeled conjugates for indicated times. (D) Cultured human AML cells (MV4-11, KG1a) quickly internalize CpG(D19)-STAT3dODN (500 nM) but not unconjugated STAT3 decoy. (E-F) CpG-STAT3dODN colocalizes and directly interacts with cytoplasmic STAT3 after internalization into target cells. (E) Direct interaction between CpG(D19)-STAT3dODN^FITC and activated STAT3 as verified using FITC- and pSTAT3-specific antibodies and proximity ligation assay. RAW264.7 macrophages were incubated with 500 nM CpG(D19)-STAT3dODN^FITC for 1 hour, fixed, and permeabilized. The interaction between the oligonucleotide and pSTAT3 using in situ proximity ligation assay (PLA) with FITC- and pSTAT3-specific antibodies labeled with PLA probes. The close proximity of both molecules is indicated by cyclic polymerase reaction producing red fluorescent spots in the cytoplasm. Shown is the image representative for 1 of 3 independent experiments with similar results; scale bar, 10 μm. (F) The intracellular localization of the FITC-labeled oligonucleotide and activated STAT3 was assessed using confocal microscopy after staining with pSTAT3-specific antibodies; inlay, the enlargement of the perinuclear area; bottom, overlay of signal intensities for fluorescent channels across the cell of interest as indicated by the dotted line: green, CpG-dODN^FITC; red, pSTAT3; blue, nuclear staining with DAPI. Shown are representative images from 1 of 3 independent experiments with similar results; scale bar = 10 µm. % max, percentage of maximum; DAPI, 4,6 diamidino-2-phenylindole.