Figure 6.

Correlation between seed-dependent gene silencing activity of siRNA and chiRNA and calculated T_m of the protein-free seed duplex. Gene silencing activity was measured using relative luciferase activity in HeLa cells transfected with psiCHECK-sm and cognate siRNAs or chiRNAs at a 50 nM concentration, as shown in Figures 4 and 7. T_m of the protein-free seed region (positions 2–8) was determined using the nearest neighbor method. (A,B) All possible 7-nt seed sequences (4⁷ = 16,384) were ordered as a function of GC content and T_m values of their double-stranded counterparts with RNA (A) and DNA (B). Note that because of its definition, class I siRNA or chiRNA cannot possess more than four GCs in the seed region. Open red (A) or blue (B) circles represent combinations of target and siRNA resulting in less than 50% relative luciferase activity. Solid red (A) or blue (B) circles represent combinations of target and siRNA with little or no off-target effect (luciferase activity >50%). The horizontal line at 21–25°C (A) or 28–41°C (B) may correspond to 50% luciferase activity reduction.