Thermodynamic stability and Watson–Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect

Short interfering RNA (siRNA) may down-regulate many unintended genes whose transcripts possess complementarity to the siRNA seed region, which contains 7 nt. The capability of siRNA to induce this off-target effect was highly correlated with the calculated melting temperature or standard free-energy change for formation of protein-free seed duplex, indicating that thermodynamic stability of seed duplex formed between the seed and target is one of the major factor in determining the degree of off-target effects. Furthermore, unlike intended gene silencing (RNA interference), off-target effect was completely abolished by introduction of a G:U pair into the seed duplex, and this loss in activity was completely recovered by a second mutation regenerating Watson–Crick pairing, indicating that seed duplex Watson–Crick pairing is also essential for off-target gene silencing. The off-target effect was more sensitive to siRNA concentration compared to intended gene silencing, which requires a near perfect sequence match between the siRNA guide strand and target mRNA.