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Designing Efficient and Specific Endoribonuclease-Prepared siRNAs

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siRNA Design

Part of the book series: Methods in Molecular Biology ((MIMB,volume 942))

Abstract

RNA interference (RNAi) has grown to be one of the main techniques for loss-of-function studies, leading to the elucidation of biological function of genes in various cellular systems and model organisms. While for many invertebrates such as Drosophila melanogaster (D. melanogaster) and Caenorhabditis elegans (C. elegans) long double-stranded RNA (dsRNA) can directly be used to induce a RNAi response, chemically synthesized small interfering RNAs (siRNAs) are typically employed in mammalian cells to avoid an interferon-like response triggered by long dsRNA (Reynolds et al., RNA 12:988–993, 2006). However, siRNAs are expensive and beset with unintentional gene targeting effects (off-targets) confounding the analysis of results from such studies. We, and others, have developed an alternative technology for RNAi in mammalian cells, termed endoribonuclease-prepared siRNA (esiRNA), which is based on the enzymatic generation of siRNA pools by digestion of long dsRNAs with recombinant RNase III in vitro (Yang et al., Proc Natl Acad Sci USA 99: 9942–9947, 2002; Myers et al., Nat Biotechnol 21:324–328; 2003). This technology has proven to be cost-efficient and reliable. Furthermore, several studies have demonstrated that complex pools of siRNAs, as inherent in esiRNAs, which target one transcript reduce off-target effects (Myers et al., J RNAi Gene Silencing 2:181, 2006; Kittler et al., Nat Methods 4:337–344, 2007). Within this chapter we describe design criteria for the generation of target-optimized esiRNAs.

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References

  1. Reynolds A et al (2006) Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA 12:988–993

    Article  CAS  Google Scholar 

  2. Yang D et al (2002) Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci USA 99:9942–9947

    Article  CAS  Google Scholar 

  3. Myers JW, Jones JT, Meyer T, Ferrell JE (2003) Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat Biotechnol 21:324–328

    Article  CAS  Google Scholar 

  4. Myers JW et al (2006) Minimizing off-target effects by using diced siRNAs for RNA interference. J RNAi Gene Silencing 2:181

    CAS  Google Scholar 

  5. Kittler R et al (2007) Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nat Methods 4:337–344

    CAS  Google Scholar 

  6. Obbard DJ et al (2009) The evolution of RNAi as a defence against viruses and transposable elements. Philos Trans R Soc Lond B Biol Sci 364:99

    Article  CAS  Google Scholar 

  7. Carrington JC, Ambros V (2003) Role of microRNAs in plant and animal development. Science 301:336–338

    Article  CAS  Google Scholar 

  8. Carpenter AE, Sabatini DM (2004) Systematic genome-wide screens of gene function. Nat Rev Genet 5:11–22

    Article  CAS  Google Scholar 

  9. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366

    Article  CAS  Google Scholar 

  10. Hutvagner G (2001) A cellular function for the RNA-interference enzyme dicer in the maturation of the let-7 small temporal RNA. Science 293:834–838

    Article  CAS  Google Scholar 

  11. Rana TM (2007) Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol 8:23–36

    Article  CAS  Google Scholar 

  12. Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200

    Article  CAS  Google Scholar 

  13. Elbashir SM et al (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498

    Article  CAS  Google Scholar 

  14. Jackson AL et al (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21:635–637

    Article  CAS  Google Scholar 

  15. Birmingham A et al (2006) 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 3:199–204

    Article  CAS  Google Scholar 

  16. Reynolds A et al (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330

    Article  CAS  Google Scholar 

  17. Chiu Y-L, Rana TM (2003) siRNA function in RNAi: a chemical modification analysis. RNA 9:1034–1048

    Article  CAS  Google Scholar 

  18. Selinger CI, Day CJ, Morrison NA (2005) Optimized transfection of diced siRNA into mature primary human osteoclasts: inhibition of cathepsin K mediated bone resorption by siRNA. J Cell Biochem 96:996–1002

    Article  CAS  Google Scholar 

  19. Kittler R et al (2007) Genome-scale RNAi profiling of cell division in human tissue culture cells. Nat Cell Biol 9:1401–1412

    Article  CAS  Google Scholar 

  20. Collinet C et al (2010) Systems survey of endocytosis by multiparametric image analysis. Nature 464:243–249

    Article  CAS  Google Scholar 

  21. Fazzio TG, Huff JT, Panning B (2008) An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell 134:162–174

    Article  CAS  Google Scholar 

  22. Galvez T et al (2007) siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a primary regulator of transferrin uptake. Genome Biol 8:R142

    Article  Google Scholar 

  23. Krastev DB et al (2011) A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly. Nat Cell Biol 13:809–818

    Article  CAS  Google Scholar 

  24. Słabicki M et al (2010) A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. PLoS Biol 8:e1000408

    Article  Google Scholar 

  25. Theis M et al (2009) Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division. EMBO J 28:1453–1465

    Article  CAS  Google Scholar 

  26. Leushacke M et al (2011) An RNA interference phenotypic screen identifies a role for FGF signals in colon cancer progression. PLoS One 6:e23381

    Article  CAS  Google Scholar 

  27. Ding L et al (2009) A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity. Cell Stem Cell 4:403–415

    Article  CAS  Google Scholar 

  28. Kittler R et al (2004) An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432:1036–1040

    Article  CAS  Google Scholar 

  29. Sontheimer EJ (2005) Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol 6:127–138

    Article  CAS  Google Scholar 

  30. Tijsterman M, Plasterk RHA (2004) Dicers at RISC; the mechanism of RNAi. Cell 117:1–3

    Article  CAS  Google Scholar 

  31. Mittal V (2004) Improving the efficiency of RNA interference in mammals. Nat Rev Genet 5:355–365

    Article  CAS  Google Scholar 

  32. Matveeva O et al (2007) Comparison of approaches for rational siRNA design leading to a new efficient and transparent method. Nucleic Acids Res 35:e63

    Article  Google Scholar 

  33. Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209–216

    Article  CAS  Google Scholar 

  34. Hardin CC, Watson T, Corregan M, Bailey C (1992) Cation-dependent transition between the quadruplex and Watson-Crick hairpin forms of d(CGCG3GCG). Biochemistry 31:833–841

    Article  CAS  Google Scholar 

  35. Geiduschek EP, Kassavetis GA (2001) The RNA polymerase III transcription apparatus. J Mol Biol 310:1–26

    Article  CAS  Google Scholar 

  36. Jagla B et al (2005) Sequence characteristics of functional siRNAs. RNA 11:864–872

    Article  CAS  Google Scholar 

  37. Shabalina SA, Spiridonov AN, Ogurtsov AY (2006) Computational models with thermodynamic and composition features improve siRNA design. BMC Bioinformatics 7:65

    Article  Google Scholar 

  38. Amarzguioui M, Prydz H (2004) An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun 316:1050–1058

    Article  CAS  Google Scholar 

  39. Anderson EM et al (2008) Experimental validation of the importance of seed complement frequency to siRNA specificity. RNA 14:853–861

    Article  CAS  Google Scholar 

  40. Manber U, Myers G (1990) In: Proceedings of the first annual ACM-SIAM symposium on discrete algorithms, SODA ‘90. Society for Industrial and Applied Mathematics, pp 319–327

    Google Scholar 

  41. Henschel A, Buchholz F, Habermann B (2004) DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Res 32:W113–W120

    Article  CAS  Google Scholar 

  42. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

    Article  Google Scholar 

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Author information

Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Vineeth Surendranath & Mirko Theis

  2. Max Planck Institute for Biology of Ageing, Cologne, Germany

    Bianca H. Habermann

  3. Department of Medical Systems Biology, University Cancer Center, University Hospital and Medical Faculty Carl Gustav Carus, University of Technology Dresden, Dresden, Germany

    Frank Buchholz

Authors
  1. Vineeth Surendranath
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  2. Mirko Theis
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  3. Bianca H. Habermann
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  4. Frank Buchholz
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Corresponding author

Correspondence to Frank Buchholz .

Editor information

Editors and Affiliations

  1. , Lineberger Comprehensive Cancer Center, University of North Carolina, West Drive 450, Chapel Hill, 27599, North Carolina, USA

    Debra J. Taxman

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Surendranath, V., Theis, M., Habermann, B.H., Buchholz, F. (2013). Designing Efficient and Specific Endoribonuclease-Prepared siRNAs. In: Taxman, D. (eds) siRNA Design. Methods in Molecular Biology, vol 942. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-119-6_11

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  • DOI: https://doi.org/10.1007/978-1-62703-119-6_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-118-9

  • Online ISBN: 978-1-62703-119-6

  • eBook Packages: Springer Protocols

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