siDirect selects siRNAs with lower Tm value at the seed region, which contains 7 nucleotides at positions 2-8 from 5′ end of the guide strand.
siRNAs down-regulate many unintended genes whose transcripts have complementarities to the siRNA seed region. The capability of siRNA to induce this seed-dependent off-target effect is highly correlated with the thermodynamic stability of the duplex formed between the seed region of the siRNA guide strand and its target mRNA. The melting temperature (Tm) for the formation of the seed duplex showed strong correlation with seed-dependent off-target effect.
The figure above shows correlation between seed-dependent off-target activity and seed Tm value. All possible 7-nt seed sequences (4^7 = 16,384) were ordered as a function of GC content and Tm. Blue: pairs of target and siRNA, giving significant off-target effects. Red: pairs of target and siRNA, with little or no off-target effects. See the reference (Ui-Tei et al., 2008) for detail.