Figure 2

Characterization of protein tags with varying lengths of polyhistidine and the siRNA-aptamer chimera.

(a) SDS-PAGE analysis of protein tags composed of a dsRBD binding domain and polyhistidines at the two termini (total number of His: 6, 18, 24 and 30), in reference to protein ladder shown to the left. Motility patterns of the four protein tags are in agreement with their calculated molecular weights of 22.6 kDa (His₆), 24.8 kDa (His₁₈), 25.8 kDa (His₂₄) and 26.8 kDa (His₃₀). (b) Characterization of dsRNA binding capability of the four protein tags with agarose gel electrophoresis. Chimera labeled with fluorophore (FAM) was incubated with the protein tags at protein/chimera molar ratios of 1, 2, or 4 for 1 h at 4°C. The dsRNA binding capability of dsRBD-His₁₈ is well preserved compared to the original dsRBD-His₆, whereas dsRBD-His₂₄ and dsRBD-His₃₀ completely lose dsRNA binding activity. (c) Evaluation of targeting specificity of the aptamer block in chimera. PSMA-positive LNCaP cells and PSMA-negative PC3 cells are treated with complex of Cy3-labeled chimera and dsRBD-His₁₈ for 12 h. Fluorescence microscopy reveals selective binding of the complex to LNCaP cells, but not PC3 cells. Scale bar: 50 μm. (d) Evaluation of silencing functionality of the siRNA block. The chimera and conventional siRNA targeting GFP (positive control) are transfected into GFP-expressing C4-2 prostate cancer cells using Lipofectamine. The silencing effect of the chimera is indistinguishable with the positive control. Scale bar: 250 μm.