siRNA Design Principles and Off-Target Effects

  • Sebastian Petri
  • Gunter Meister
Part of the Methods in Molecular Biology book series (MIMB, volume 986)


Short interfering RNAs (siRNAs) are a major research tool that allows for knock-down of target genes via selective mRNA destruction in almost all eukaryotic organisms. siRNAs typically consist of a synthetic ∼21 nucleotide (nt) RNA-duplex where one strand is designed with perfect complementarity to the target mRNA. Although siRNAs were initially thought to be very target-specific because of their design, it turned out during the last years that all siRNAs have a more or less pronounced intrinsic off-target activity which can make the interpretation of data from siRNA experiments difficult. Here we describe essential rules for siRNA design that should be taken into account in order to obtain potent siRNAs with minimal off-target activity. In addition, we describe how to control for off-target activity in siRNA experiments.

Key words

siRNA Argonaute proteins siRNA off-target activity siRNA on-target activity siRNA design siRNA modifications 



Our research is supported in part by the BMBF (NGFN+, FKZ PIM-01GS0804-5 to G.M.), the Bavarian Genome Research Network (BayGene to G.M.), the Deutsche Forschungsgemein­schaft (DFG), and Roche Kulmbach GmbH. S.P. received a fellowship from the Roche Postdoc Fellowship Program.


  1. 1.
    Fire A, Xu S, Montgomery M et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811PubMedCrossRefGoogle Scholar
  2. 2.
    Tuschl T (2001) RNA interference and small interfering RNAs. ChemBioChem 2:239–245PubMedCrossRefGoogle Scholar
  3. 3.
    Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349PubMedCrossRefGoogle Scholar
  4. 4.
    Elbashir SM, Harborth J, Lendeckel W et al (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature 411:494–498PubMedCrossRefGoogle Scholar
  5. 5.
    Dorsett Y, Tuschl T (2004) siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 3:318–329PubMedCrossRefGoogle Scholar
  6. 6.
    Kim DH, Rossi JJ (2007) Strategies for silencing human disease using RNA interference. Nat Rev Genet 8:173–184PubMedCrossRefGoogle Scholar
  7. 7.
    Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10:126–139PubMedCrossRefGoogle Scholar
  8. 8.
    Siomi H, Siomi MC (2009) On the road to reading the RNA-interference code. Nature 457:396–404PubMedCrossRefGoogle Scholar
  9. 9.
    Filipowicz W, Jaskiewicz L, Kolb FA et al (2005) Post-transcriptional gene silencing by siRNAs and miRNAs. Curr Opin Struct Biol 15:331–341PubMedCrossRefGoogle Scholar
  10. 10.
    Hutvagner G, Simard MJ (2008) Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol 9:22–32PubMedCrossRefGoogle Scholar
  11. 11.
    Ender C, Meister G (2010) Argonaute proteins at a glance. J Cell Sci 123:1819–1823PubMedCrossRefGoogle Scholar
  12. 12.
    Peters L, Meister G (2007) Argonaute proteins: mediators of RNA silencing. Mol Cell 26:611–623PubMedCrossRefGoogle Scholar
  13. 13.
    Tolia NH, Joshua-Tor L (2007) Slicer and the argonautes. Nat Chem Biol 3:36–43PubMedCrossRefGoogle Scholar
  14. 14.
    Jinek M, Doudna JA (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457:405–412PubMedCrossRefGoogle Scholar
  15. 15.
    Carmell MA, Xuan Z, Zhang MQ et al (2002) The argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev 16:2733–2742PubMedCrossRefGoogle Scholar
  16. 16.
    Meister G, Landthaler M, Patkaniowska A et al (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 15:185–197PubMedCrossRefGoogle Scholar
  17. 17.
    Liu J, Carmell MA, Rivas FV et al (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437–1441PubMedCrossRefGoogle Scholar
  18. 18.
    Wu L, Fan J, Belasco JG (2008) Importance of translation and nonnucleolytic ago proteins for on-target RNA interference. Curr Biol 18:1327–1332PubMedCrossRefGoogle Scholar
  19. 19.
    Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17:438–442PubMedCrossRefGoogle Scholar
  20. 20.
    Jackson AL, Linsley PS (2010) Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov 9:57–67PubMedCrossRefGoogle Scholar
  21. 21.
    Semizarov D, Frost L, Sarthy A et al (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci USA 100:6347–6352PubMedCrossRefGoogle Scholar
  22. 22.
    Persengiev SP, Zhu X, Green MR (2004) Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10:12–18PubMedCrossRefGoogle Scholar
  23. 23.
    Jackson AL, Bartz SR, Schelter J et al (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21:635–637PubMedCrossRefGoogle Scholar
  24. 24.
    Scacheri PC, Rozenblatt-Rosen O, Caplen NJ et al (2004) Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc Natl Acad Sci USA 101:1892–1897PubMedCrossRefGoogle Scholar
  25. 25.
    Birmingham A, Anderson EM, Reynolds A et al (2006) 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 3:199–204PubMedCrossRefGoogle Scholar
  26. 26.
    Jackson AL, Burchard J, Schelter J et al (2006) Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 12:1179–1187PubMedCrossRefGoogle Scholar
  27. 27.
    Lin X, Ruan X, Anderson MG et al (2005) siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res 33:4527–4535PubMedCrossRefGoogle Scholar
  28. 28.
    Grimm D, Streetz KL, Jopling CL et al (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537–541PubMedCrossRefGoogle Scholar
  29. 29.
    Khan AA, Betel D, Miller ML et al (2009) Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs. Nat Biotechnol 27:549–555PubMedCrossRefGoogle Scholar
  30. 30.
    Fedorov Y, Anderson EM, Birmingham A et al (2006) Off-target effects by siRNA can induce toxic phenotype. RNA 12:1188–1196PubMedCrossRefGoogle Scholar
  31. 31.
    Hornung V, Guenthner-Biller M, Bourquin C et al (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med 11:263–270PubMedCrossRefGoogle Scholar
  32. 32.
    Judge AD, Sood V, Shaw JR et al (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 23:457–462PubMedCrossRefGoogle Scholar
  33. 33.
    Chaudhary A, Srivastava S, Garg S (2011) Development of a software tool and criteria evaluation for efficient design of small interfering RNA. Biochem Biophys Res Commun 404:313–320PubMedCrossRefGoogle Scholar
  34. 34.
    Wang X, Varma RK, Beauchamp L et al (2009) Selection of hyperfunctional siRNAs with improved potency and specificity. Nucleic Acids Res 37:e152PubMedCrossRefGoogle Scholar
  35. 35.
    Tafer H, Ameres SL, Obernosterer G et al (2008) The impact of target site accessibility on the design of effective siRNAs. Nat Biotechnol 26:578–583PubMedCrossRefGoogle Scholar
  36. 36.
    Ui-Tei K, Naito Y, Takahashi F et al (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32:936–948PubMedCrossRefGoogle Scholar
  37. 37.
    Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209–216PubMedCrossRefGoogle Scholar
  38. 38.
    Schwarz DS, Hutvágner G, Du T, Xu Z et al (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208PubMedCrossRefGoogle Scholar
  39. 39.
    Parker JS, Roe SM, Barford D (2005) Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex. Nature 434:663–666PubMedCrossRefGoogle Scholar
  40. 40.
    Ma JB, Yuan YR, Meister G et al (2005) Structural basis for 5’-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434:666–670PubMedCrossRefGoogle Scholar
  41. 41.
    Frank F, Sonenberg N, Nagar B (2010) Structural basis for 5’-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 465:818–822PubMedCrossRefGoogle Scholar
  42. 42.
    Allerson CR, Sioufi N, Jarres R et al (2005) Fully 2’-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J Med Chem 48:901–904PubMedCrossRefGoogle Scholar
  43. 43.
    Addepalli H, Meena, Peng CG et al (2010) Modulation of thermal stability can enhance the potency of siRNA. Nucleic Acids Res 38:7320–7331Google Scholar
  44. 44.
    Petri S, Dueck A, Lehmann G et al (2011) Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects. RNA 17:737–749PubMedCrossRefGoogle Scholar
  45. 45.
    Bramsen JB, Pakula MM, Hansen TB et al (2010) A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects. Nucleic Acids Res 38:5761–5773PubMedCrossRefGoogle Scholar
  46. 46.
    Bramsen JB, Laursen MB, Damgaard CK et al (2007) Improved silencing properties using small internally segmented interfering RNAs. Nucleic Acids Res 35:5886–5897PubMedCrossRefGoogle Scholar
  47. 47.
    Chen PY, Weinmann L, Gaidatzis D et al (2008) Strand-specific 5’-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity. RNA 14:263–274PubMedCrossRefGoogle Scholar
  48. 48.
    Soutschek J, Akinc A, Bramlage B et al (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432:173–178PubMedCrossRefGoogle Scholar
  49. 49.
    Bramsen JB, Kjems J (2011) Chemical modification of small interfering RNA. Methods Mol Biol 721:77–103PubMedCrossRefGoogle Scholar
  50. 50.
    Caffrey DR, Zhao J, Song Z et al (2011) siRNA off-target effects can be reduced at concentrations that match their individual potency. PLoS One 6:e21503Google Scholar
  51. 51.
    Grunweller A, Wyszko E, Bieber B et al (2003) Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2’-O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Res 31:3185–3193PubMedCrossRefGoogle Scholar
  52. 52.
    Vickers TA, Lima WF, Nichols JG et al (2007) Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells. Nucleic Acids Res 35:6598–6610PubMedCrossRefGoogle Scholar
  53. 53.
    Diederichs S, Jung S, Rothenberg SM et al (2008) Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites. Proc Natl Acad Sci USA 105:9284–9289PubMedCrossRefGoogle Scholar

Copyright information

© SpringerScience+Business Media New York 2013

Authors and Affiliations

  • Sebastian Petri
    • 1
  • Gunter Meister
    • 1
  1. 1.Laboratory for RNA Biology, Biochemistry Center Regensburg (BZR)University of RegensburgRegensburgGermany

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