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

Ui-Tei K, Naito Y, Nishi K, Juni A, Saigo K - Nucleic Acids Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan. ktei@biochem.s.u-tokyo.ac.jp

ABSTRACT
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.

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Correlation between seed-dependent gene-silencing activity and calculated Tm 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. Tm 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 Tm 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) Tm distribution of 733 human microRNAs registered in miRbase. Ordinate represents calculated seed-duplex Tm. Abscissa represents percentage. Seventy-five percent of 565 siRNAs registered was estimated to be associated with Tm more than 21.5°C.
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Figure 3: Correlation between seed-dependent gene-silencing activity and calculated Tm 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. Tm 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 Tm 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) Tm distribution of 733 human microRNAs registered in miRbase. Ordinate represents calculated seed-duplex Tm. Abscissa represents percentage. Seventy-five percent of 565 siRNAs registered was estimated to be associated with Tm more than 21.5°C.

Mentions: A considerable deviation was observed in luc activity measurements shown in Figure 2B and C. This may be due in part to differences in the non-seed sequence and/or its counterpart in target mRNA, since the target sequences that correspond to the non-seed region make an appreciable contribution to target recognition by miRNAs and/or siRNAs in microarray profiling (1,6,8) (Supplementary Figure S1). The reporter assay using eight different targets with common seed and different non-seed sequences showed that off-target effects are rather various, indicating possible involvement of siRNA non-seed region and/or its target counterpart in off-target effect (Supplementary Figure S3). However, correlation between seed-dependent gene silencing activity of siRNA used in this study (Figure 1C) and calculated Tm of protein-free seed duplex of them aligned in all possible 7-nt sequences (47 = 16 384) may suggest that 21.5°C serves as a kind of benchmark Tm (Figure 3A), which may discriminate almost off-target-free seed sequences from off-target-positive ones.Figure 3.


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

Ui-Tei K, Naito Y, Nishi K, Juni A, Saigo K - Nucleic Acids Res. (2008)

Correlation between seed-dependent gene-silencing activity and calculated Tm 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. Tm 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 Tm 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) Tm distribution of 733 human microRNAs registered in miRbase. Ordinate represents calculated seed-duplex Tm. Abscissa represents percentage. Seventy-five percent of 565 siRNAs registered was estimated to be associated with Tm more than 21.5°C.
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Related In: Results  -  Collection

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Figure 3: Correlation between seed-dependent gene-silencing activity and calculated Tm 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. Tm 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 Tm 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) Tm distribution of 733 human microRNAs registered in miRbase. Ordinate represents calculated seed-duplex Tm. Abscissa represents percentage. Seventy-five percent of 565 siRNAs registered was estimated to be associated with Tm more than 21.5°C.
Mentions: A considerable deviation was observed in luc activity measurements shown in Figure 2B and C. This may be due in part to differences in the non-seed sequence and/or its counterpart in target mRNA, since the target sequences that correspond to the non-seed region make an appreciable contribution to target recognition by miRNAs and/or siRNAs in microarray profiling (1,6,8) (Supplementary Figure S1). The reporter assay using eight different targets with common seed and different non-seed sequences showed that off-target effects are rather various, indicating possible involvement of siRNA non-seed region and/or its target counterpart in off-target effect (Supplementary Figure S3). However, correlation between seed-dependent gene silencing activity of siRNA used in this study (Figure 1C) and calculated Tm of protein-free seed duplex of them aligned in all possible 7-nt sequences (47 = 16 384) may suggest that 21.5°C serves as a kind of benchmark Tm (Figure 3A), which may discriminate almost off-target-free seed sequences from off-target-positive ones.Figure 3.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan. ktei@biochem.s.u-tokyo.ac.jp

ABSTRACT
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.

Show MeSH