Home LiteratureArticle Details
PMID: 21367974 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects.

RNA (New York, N.Y.) ·Vol. 17 ·No. 4 ·2011-04-00 ·Pages 737-49

Petri S, Dueck A, Lehmann G, Putz N, Rüdel S, Kremmer E, Meister G

Abstract

Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.

MeSH Terms
Argonaute Proteins Eukaryotic Initiation Factor-2/genetics,metabolism Eukaryotic Initiation Factors/genetics,metabolism HEK293 Cells Humans RNA Stability RNA, Small Interfering/chemistry,metabolism RNA-Induced Silencing Complex/chemistry,metabolism
Chemicals
AGO1 protein, human AGO2 protein, human AGO3 protein, human AGO4 protein, human Argonaute Proteins Eukaryotic Initiation Factor-2 Eukaryotic Initiation Factors RNA, Small Interfering RNA-Induced Silencing Complex
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Petri Sebastian
University of Regensburg, 93053 Regensburg, Germany.
Dueck Anne
Lehmann Gerhard
Putz Nicholas
Rüdel Sabine
Kremmer Elisabeth
Meister Gunter
References (46)
46 references, click to expand
  1. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation.
    Cell. 2005 Nov 18;123(4):621-9 PMID: 16271385
  2. Mammalian microRNAs predominantly act to decrease target mRNA levels.
    Nature. 2010 Aug 12;466(7308):835-40 PMID: 20703300
  3. Sorting of Drosophila small silencing RNAs.
    Cell. 2007 Jul 27;130(2):299-308 PMID: 17662944
  4. Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity.
    RNA. 2008 Feb;14(2):263-74 PMID: 18094121
  5. Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity.
    RNA. 2006 Jul;12(7):1179-87 PMID: 16682560
  6. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?
    Nat Rev Genet. 2008 Feb;9(2):102-14 PMID: 18197166
  7. Functional siRNAs and miRNAs exhibit strand bias.
    Cell. 2003 Oct 17;115(2):209-16 PMID: 14567918
  8. Identification of human microRNA targets from isolated argonaute protein complexes.
    RNA Biol. 2007 Jun;4(2):76-84 PMID: 17637574
  9. Mechanisms of gene silencing by double-stranded RNA.
    Nature. 2004 Sep 16;431(7006):343-9 PMID: 15372041
  10. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.
    Mol Cell. 2004 Jul 23;15(2):185-97 PMID: 15260970
  11. Drosophila microRNAs are sorted into functionally distinct argonaute complexes after production by dicer-1.
    Cell. 2007 Jul 27;130(2):287-97 PMID: 17662943
  12. Comparison of siRNA-induced off-target RNA and protein effects.
    RNA. 2007 Mar;13(3):385-95 PMID: 17237357
  13. Biogenesis of small RNAs in animals.
    Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39 PMID: 19165215
  14. The promises and pitfalls of RNA-interference-based therapeutics.
    Nature. 2009 Jan 22;457(7228):426-33 PMID: 19158789
  15. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity.
    Nucleic Acids Res. 2009 May;37(9):2867-81 PMID: 19282453
  16. Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites.
    Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9284-9 PMID: 18591665
  17. Structural determinants of miRNAs for RISC loading and slicer-independent unwinding.
    Nat Struct Mol Biol. 2009 Sep;16(9):953-60 PMID: 19684602
  18. A protein sensor for siRNA asymmetry.
    Science. 2004 Nov 19;306(5700):1377-80 PMID: 15550672
  19. Argonaute proteins at a glance.
    J Cell Sci. 2010 Jun 1;123(Pt 11):1819-23 PMID: 20484662
  20. Importance of translation and nonnucleolytic ago proteins for on-target RNA interference.
    Curr Biol. 2008 Sep 9;18(17):1327-32 PMID: 18771919
  21. Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs.
    Nucleic Acids Res. 2011 Mar;39(5):1823-32 PMID: 21047800
  22. Expression profiling reveals off-target gene regulation by RNAi.
    Nat Biotechnol. 2003 Jun;21(6):635-7 PMID: 12754523
  23. 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.
    Nat Methods. 2006 Mar;3(3):199-204 PMID: 16489337
  24. Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 'Slicer' independent and can be mediated by Ago1.
    Nucleic Acids Res. 2009 Nov;37(20):6927-41 PMID: 19767612
  25. Improved silencing properties using small internally segmented interfering RNAs.
    Nucleic Acids Res. 2007;35(17):5886-97 PMID: 17726057
  26. Off-targeting and other non-specific effects of RNAi experiments in mammalian cells.
    Curr Opin Mol Ther. 2007 Jun;9(3):248-57 PMID: 17608023
  27. Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes.
    RNA. 2007 Jan;13(1):22-9 PMID: 17123955
  28. siRNAs: applications in functional genomics and potential as therapeutics.
    Nat Rev Drug Discov. 2004 Apr;3(4):318-29 PMID: 15060527
  29. A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects.
    Nucleic Acids Res. 2010 Sep;38(17):5761-73 PMID: 20453030
  30. A multifunctional human Argonaute2-specific monoclonal antibody.
    RNA. 2008 Jun;14(6):1244-53 PMID: 18430891
  31. A human snoRNA with microRNA-like functions.
    Mol Cell. 2008 Nov 21;32(4):519-28 PMID: 19026782
  32. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.
    Cell. 2005 Nov 18;123(4):607-20 PMID: 16271386
  33. Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells.
    EMBO Rep. 2007 Nov;8(11):1052-60 PMID: 17932509
  34. Distinct passenger strand and mRNA cleavage activities of human Argonaute proteins.
    Nat Struct Mol Biol. 2009 Dec;16(12):1259-66 PMID: 19946268
  35. Slicer function of Drosophila Argonautes and its involvement in RISC formation.
    Genes Dev. 2005 Dec 1;19(23):2837-48 PMID: 16287716
  36. Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing.
    RNA. 2006 Jul;12(7):1197-205 PMID: 16682562
  37. Getting to the root of miRNA-mediated gene silencing.
    Cell. 2008 Jan 11;132(1):9-14 PMID: 18191211
  38. Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo.
    Mol Biosyst. 2010 May;6(5):862-70 PMID: 20567772
  39. A three-dimensional view of the molecular machinery of RNA interference.
    Nature. 2009 Jan 22;457(7228):405-12 PMID: 19158786
  40. Argonaute2 is the catalytic engine of mammalian RNAi.
    Science. 2004 Sep 3;305(5689):1437-41 PMID: 15284456
  41. Asymmetry in the assembly of the RNAi enzyme complex.
    Cell. 2003 Oct 17;115(2):199-208 PMID: 14567917
  42. Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells.
    Nucleic Acids Res. 2007;35(19):6598-610 PMID: 17905815
  43. Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.
    Cell. 2009 Feb 6;136(3):496-507 PMID: 19167051
  44. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
    Nucleic Acids Res. 2005 Aug 09;33(14):4527-35 PMID: 16091630
  45. 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
  46. Cleavage of the siRNA passenger strand during RISC assembly in human cells.
    EMBO Rep. 2006 Mar;7(3):314-20 PMID: 16439995
Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1469-9001
DOI
10.1261/rna.2348111
Published
2011-04-00
Epub
2011-00-02
Pages
737-49
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC3062184
Subset
IM
External Links
PubMed source DOI: 10.1261/rna.2348111 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