EpApt-siEp fabrication, <i>in vitro</i> processing by dicer enzyme, cell surface binding and internalization.

<p><b>A</b>. EpCAM aptamer secondary structure prediction from Mfold online. <b>B</b>. EpCAM aptamer siRNA chimeric construct carrying the siRNA targeting EpCAM (EpApt-siEp) is folded using Mfold online and the aptamer is indicated in blue box and the siRNA inside red box. <b>C</b>. EpCAM aptamer siRNA chimeric construct was incubated with the recombinant dicer enzyme at 37˚C for 18h. The reactions were performed without dicer as control reaction. Polyacrylamide gel electrophoresis of the reactions with and without dicer enzyme were run on 15% gel and stained with EtBr. The processed 21bp siRNA and unprocessed construct were observed. <b>D</b>. EpCAM aptamer siRNA chimeric construct was added to WERI-Rb1 and MCF7 cells in binding buffer and analyzed by flow cytometry. The overlay graph shows the uptake of the chimeric aptamer. <b>E</b>. Scatter plot showing the uptake of EpApt-siEp by the RB cell line, WERI-Rb1 and RB primary tumor cells. <b>F</b>. EpCAM aptamer siRNA chimeric construct was added to primary RB cells in media without serum for 2hr at 37˚C followed by washing with 1X PBS. Microscopic images were taken at 20X objective under phase and FITC channels of control cells alone and cells with EpApt-siEp. Data represents mean ± SD. Experiments were repeated 3 times independently with similar results. **P value of 0.01–0.001; *P value of 0.05–0.01.