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.
Ui-Tei
Kumiko
K
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan. ktei@biochem.s.u-tokyo.ac.jp
Naito
Yuki
Y
Nishi
Kenji
K
Juni
Aya
A
Saigo
Kaoru
K
D020029:Base Pairing; D001483:Base Sequence; D023281:Genomics; D006147:Guanine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000737:chemistry*; D013816:Thermodynamics; D014498:Uracil / Q000737:chemistry
D034741:RNA, Small Interfering; D014498:Uracil; D006147:Guanine