siDirect selects siRNAs with lower Tm values at the seed region, which contains 7 nucleotides at positions 2-8 from the 5′ end of the guide strand.
siRNAs down-regulate many unintended genes whose transcripts have complementarities to the siRNA seed region. The capability of siRNAs 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 effects.
The figure above shows the correlation between seed-dependent off-target activity and the seed Tm value. All possible 7-nt seed sequences (4^7 = 16,384) were ordered based on their GC content and Tm. Blue: pairs of siRNA and the target, giving significant off-target effects. Red: pairs of siRNA and the target, with little or no off-target effects. See the reference (Ui-Tei et al., 2008) for detail.