Figure 3.

Correlation between seed-dependent gene-silencing activity and calculated T_m of protein-free seed duplex. Gene-silencing activity was measured using relative luc activity in HeLa cells transfected with psiCHECK-sm and cognate siRNAs at 50 nM as shown in Figure 1C. T_m of the protein-free seed region (positions 2–8) was determined using nearest neighbor method (Figure 2C). (A) All possible 7-nt seed sequences (4⁷ = 16 384) were ordered as a function of GC content and T_m values of their ds counterparts. Note that, because of its definition, class I siRNA cannot possess more than four GC in the seed region. Blue: combinations of target and siRNA, giving less than 50% relative luc activity. Red: combinations of target and siRNA with little or no off-target effect (luc activity >50%). Dotted line at 21.5°C may correspond to 50% luc activity reduction. (B) T_m distribution of 733 human microRNAs registered in miRbase. Ordinate represents calculated seed-duplex T_m. Abscissa represents percentage. Seventy-five percent of 565 siRNAs registered was estimated to be associated with T_m more than 21.5°C.