Figure 4.

Effects of 5′-2′-OMe modifications on silencing, guide-strand incorporation, strand separation, asymmetry, and RISC-target interaction. (A) ER293 cells were transfected with the indicated amounts of differently modified siTK3 together with the fixed concentration of the pGL2-Control and pRL-TK reporter plasmids. After 48 h, the ratios of target to control luciferase concentration were normalized to the NegsiRNA control (indicated in black). siTK3-3′4nt-as/ss, light gray; siTK3-5′2-3′4nt-as/ss, lined bar; siTK3-as3′4nt/ss5′2-3′4nt, dark gray; siTK3-as5′2-3′4nt/ss3′4nt, open bar. The plotted data were averaged from six independent experiments ± SD. (B) Cross-correlation amplitudes in the cytoplasm (solid bars) and nucleus (open bars) after 3 and 12 h of incubation for the indicated siRNAs (mean ± SE). (C) Target interaction levels with EGFP-RISC determined by FCCS in living cells. Cross-correlation measurements were performed 1, 2, or 3 h after microinjection of labeled target RNA. Data are represented as the mean ± SE; solid and open bars indicate measurements obtained in the cytoplasm and nucleus, respectively. (D) The levels of target RNA bound to EGFP-Ago2 are plotted against the silencing values obtained for the siRNAs at 1 nM concentration after 48 h: 1 h, black diamonds; 2 h, open squares; 3 h, open triangles.