Home LiteratureArticle Details
PMID: 18988625 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Nucleic acids research ·Vol. 36 ·No. 22 ·2008-12-00 ·Pages 7100-9

Ui-Tei K, Naito Y, Nishi K, Juni A, Saigo K

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.

MeSH Terms
Base Pairing Base Sequence Genomics Guanine/chemistry HeLa Cells Humans RNA Interference RNA Stability RNA, Small Interfering/chemistry Thermodynamics Uracil/chemistry
Chemicals
RNA, Small Interfering Uracil Guanine
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Ui-Tei Kumiko
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan. ktei@biochem.s.u-tokyo.ac.jp
Naito Yuki
Nishi Kenji
Juni Aya
Saigo Kaoru
References (27)
27 references, click to expand
  1. MicroRNA targeting specificity in mammals: determinants beyond seed pairing.
    Mol Cell. 2007 Jul 6;27(1):91-105 PMID: 17612493
  2. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation.
    Nature. 2008 Oct 23;455(7216):1124-8 PMID: 18806776
  3. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.
    Cell. 2002 Sep 6;110(5):563-74 PMID: 12230974
  4. Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways.
    Mol Cell. 2002 Sep;10(3):537-48 PMID: 12408822
  5. Robust estimators for expression analysis.
    Bioinformatics. 2002 Dec;18(12):1585-92 PMID: 12490442
  6. Expression profiling reveals off-target gene regulation by RNAi.
    Nat Biotechnol. 2003 Jun;21(6):635-7 PMID: 12754523
  7. Asymmetry in the assembly of the RNAi enzyme complex.
    Cell. 2003 Oct 17;115(2):199-208 PMID: 14567917
  8. Functional siRNAs and miRNAs exhibit strand bias.
    Cell. 2003 Oct 17;115(2):209-16 PMID: 14567918
  9. MicroRNAs: genomics, biogenesis, mechanism, and function.
    Cell. 2004 Jan 23;116(2):281-97 PMID: 14744438
  10. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.
    Nucleic Acids Res. 2004;32(3):936-48 PMID: 14769950
  11. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells.
    Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7 PMID: 14769924
  12. Improved free-energy parameters for predictions of RNA duplex stability.
    Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373-7 PMID: 2432595
  13. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
    Nucleic Acids Res. 2005;33(14):4527-35 PMID: 16091630
  14. 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.
    Nat Methods. 2006 Mar;3(3):199-204 PMID: 16489337
  15. Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity.
    RNA. 2006 Jul;12(7):1179-87 PMID: 16682560
  16. Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing.
    RNA. 2006 Jul;12(7):1197-205 PMID: 16682562
  17. Thermodynamics of base pairing.
    Curr Opin Struct Biol. 1996 Jun;6(3):299-304 PMID: 8804832
  18. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
    Cell. 2005 Jan 14;120(1):15-20 PMID: 15652477
  19. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.
    Nature. 2005 Feb 17;433(7027):769-73 PMID: 15685193
  20. Comparison of different melting temperature calculation methods for short DNA sequences.
    Bioinformatics. 2005 Mar15;21(6):711-22 PMID: 15501913
  21. Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein.
    Nature. 2005 Mar 31;434(7033):666-70 PMID: 15800629
  22. RNAi: the nuts and bolts of the RISC machine.
    Cell. 2005 Jul 15;122(1):17-20 PMID: 16009129
  23. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage.
    Mol Cell. 2005 Aug 5;19(3):405-19 PMID: 16061186
  24. miRBase: tools for microRNA genomics.
    Nucleic Acids Res. 2008 Jan;36(Database issue):D154-8 PMID: 17991681
  25. Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.
    Nucleic Acids Res. 2008 Apr;36(7):2136-51 PMID: 18267968
  26. miR-148 targets human DNMT3b protein coding region.
    RNA. 2008 May;14(5):872-7 PMID: 18367714
  27. The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems.
    EMBO Rep. 2000 Jul;1(1):18-23 PMID: 11256617
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkn902
Email
ktei@biochem.s.u-tokyo.ac.jp
Published
2008-12-00
Epub
2008-00-06
Pages
7100-9
Language
English
Region
England
NLM ID
0411011
PMCID
PMC2602766
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/gkn902 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2