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Designing Optimal siRNA Based on Target Site Accessibility

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 623)

Abstract

RNA interference, mediated by small interfering RNAs (siRNAs), is a powerful tool for investigation of gene functions and it is increasingly being used as a therapeutic agent. However, not all siRNAs are equally potent - although simple rules for the selection of good siRNAs were proposed early on, siRNAs are still plagued with widely fluctuating efficiency. Recently, new design tools that incorporate both the structural features of the targeted RNAs and the sequence features of the siRNAs have substantially improved the efficacy of siRNAs. In this chapter, we present the algorithms behind these accessibility-aided tools and show how to design efficient siRNAs with their help.

Key words

RNA interference mRNA structure Target accessibility siRNA design tools Tutorial 
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References

  1. 1.
    Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811CrossRefPubMedGoogle Scholar
  2. 2.
    Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498CrossRefPubMedGoogle Scholar
  3. 3.
    Stein, C. A. (2001) Antisense that comes naturally. Nat. Biotechnol. 19, 737–738.CrossRefPubMedGoogle Scholar
  4. 4.
    Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E. and Prydz, H. (2002) Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 30, 1757–1766.CrossRefPubMedGoogle Scholar
  5. 5.
    Patzel, V. (2007) In silico selection of active siRNA. Drug Discov. Today 12, 139–148.CrossRefPubMedGoogle Scholar
  6. 6.
    Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W. and Tuschl, T. (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888.CrossRefPubMedGoogle Scholar
  7. 7.
    Khvorova, A., Reynolds, A. and Jayasena, S.D. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216.CrossRefPubMedGoogle Scholar
  8. 8.
    Schwarz, D. S., Hutvágner, G., Du, T., Xu, Z., Aronin, N. and Zamore, P.D. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208.CrossRefPubMedGoogle Scholar
  9. 9.
    Amarzguioui, M. and Prydz, H. (2004) An algorithm for selection of functional siRNA sequences. Biochem. Biophys. Res. Commun. 316, 1050–1058.CrossRefPubMedGoogle Scholar
  10. 10.
    Hohjoh, H. (2004) Enhancement of RNAi activity by improved siRNA duplexes. FEBS Lett. 557, 193–198.CrossRefPubMedGoogle Scholar
  11. 11.
    Hsieh, A.C., Bo, R., Manola, J., Vazquez, F., Bare, O., Khvorova, A., Scaringe, S. and Sellers, W.R. (2004) A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens. Nucleic Acids Res. 32, 893–901.CrossRefPubMedGoogle Scholar
  12. 12.
    Takasaki, S., Kotani, S. and Konagaya, A. (2004) An effective method for selecting siRNA target sequences in mammalian cells. Cell Cycle 3, 790–795.PubMedGoogle Scholar
  13. 13.
    Ui-Tei, K., Naito, Y., Takahashi, F. , Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R. and Saigo, K. (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 32, 936–948.CrossRefPubMedGoogle Scholar
  14. 14.
    Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S. and Khvorova, A. (2004) Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330.CrossRefPubMedGoogle Scholar
  15. 15.
    Patzel, V., Rutz S., Dietrich, I., Köberle, C., Scheffold, A. and Kaufmann, S.H.E. (2005) Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency. Nat. Biotechnol. 23, 1440–1444.CrossRefPubMedGoogle Scholar
  16. 16.
    Saetrom, P. (2004) Predicting the efficacy of short oligonucleotides in antisense and RNAi experiments with boosted genetic programming. Bioinformatics 20, 3055–3063.CrossRefPubMedGoogle Scholar
  17. 17.
    Ren, Y., Gong, W., Xu, Q., Zheng, X., Lin, D., Wang, Y., and Li, T. (2006) siRecords: an extensive database of mammalian siRNAs with efficacy ratings. Bioinformatics 22, 1027–1028.CrossRefPubMedGoogle Scholar
  18. 18.
    Huesken, D., Lange, J., Mickanin, C., Weiler, J., Asselbergs, F., Warner, J. et al. (2005) Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995–1001.CrossRefPubMedGoogle Scholar
  19. 19.
    Lima, W. F., Monia, B. P., Ecker, D. J. and Freier, S. M. (1992) Implication of RNA structure on antisense oligonucleotide hybridization kinetics. Biochemistry 31, 12055–12061.CrossRefPubMedGoogle Scholar
  20. 20.
    Vickers, T.A., Wyatt, J.R. and Freier, S.M. (2000) Effects of RNA secondary structure on cellular antisense activity. Nucleic Acids Res. 28, 1340–1347.CrossRefPubMedGoogle Scholar
  21. 21.
    Mir, K.U. and Southern E.M. (1999) Determining the influence of structure on hybridization using oligonucleotide arrays. Nat. Biotechnol. 17, 788–792.CrossRefPubMedGoogle Scholar
  22. 22.
    Milner, N., Mir, K.U., and Southern, E.M. (1997) Selecting effective antisense reagents on combinatorial oligonucleotide arrays. Nat. Biotechnol. 15, 537–541.CrossRefPubMedGoogle Scholar
  23. 23.
    Zhao, J. J., and Lemke, G. (1998) Rules for ribozymes. Mol. Cell Neurosci. 11, 92–97.CrossRefPubMedGoogle Scholar
  24. 24.
    Ding, Y., and Lawrence, C.E. (2001) Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond. Nucleic Acids Res. 29, 1034–1046.CrossRefPubMedGoogle Scholar
  25. 25.
    Bohula, E.A., Salisbury, A.J., Sohail, M., Playford, M.P., Riedemann, J., Southern, E.M. and Macaulay, V.M. (2003) The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J. Biol. Chem. 278, 15991–15997.CrossRefPubMedGoogle Scholar
  26. 26.
    Kretschmer-Kazemi Far R. and Sczakiel, G. (2003) The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res. 31, 4417–4424.CrossRefPubMedGoogle Scholar
  27. 27.
    Xu, Y., Zhang, H-Y., Thormeyer, D., Larsson, O., Du, Q., Elmén, J., Wahlestedt, C. and Liang, Z. (2003) Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs. Biochem. Biophys. Res. Commun. 306, 712–717.CrossRefPubMedGoogle Scholar
  28. 28.
    Vickers, T.A., Koo, S., Bennett, C.F., Crooke, S.T., Dean, N.M. and Baker, B.F. (2003) Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J. Biol. Chem. 278, 7108–7118.CrossRefPubMedGoogle Scholar
  29. 29.
    Ding, Y., Chan, C. Y. and Lawrence, C. E. (2004) Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res. 32 (Web Server issue), W135-W141.CrossRefPubMedGoogle Scholar
  30. 30.
    Shao, Y., Chan, C.Y., Maliyekkel, A., Lawrence, C.E., Roninson, I. B. and Ding, Y. (2007) Effect of target secondary structure on RNAi efficiency. RNA 13, 1631–1640.CrossRefPubMedGoogle Scholar
  31. 31.
    Luo, K.Q. and Chang, D.C. (2004) The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem. Biophys. Res. Commun. 318, 303–310.CrossRefPubMedGoogle Scholar
  32. 32.
    Yoshinari, K., Miyagishi, M. and Taira ,K. (2004) Effects on RNAi of the tight structure, sequence and position of the targeted region. Nucleic Acids Res. 32, 691–699.CrossRefPubMedGoogle Scholar
  33. 33.
    Overhoff, M., Alken, M., Far, R.K., Lemaitre, M., Lebleu, B., Sczakiel, G. and Robbins, I. (2005) Local RNA target structure influences siRNA efficacy: a systematic global analysis. J. Mol. Biol. 348, 871–881.CrossRefPubMedGoogle Scholar
  34. 34.
    Schubert, S., Grünweller, A., Erdmann, V.A. and Kurreck, J. (2005) Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J. Mol. Biol. 348, 883–893.CrossRefPubMedGoogle Scholar
  35. 35.
    Brown, J.R. and Sanseau, P. (2005) A computational view of microRNAs and their targets. Drug Discov. Today 10, 595–601.CrossRefPubMedGoogle Scholar
  36. 36.
    Ameres, S. L., Martinez, J. and Schroeder, R. (2007) Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130, 101–112.CrossRefPubMedGoogle Scholar
  37. 37.
    Lu, Z.J., and Mathews, D.H. (2008) OligoWalk: an online siRNA design tool utilizing hybridization thermodynamics. Nucleic Acids Res. 36 (Web Server issue), W104-W108.CrossRefPubMedGoogle Scholar
  38. 38.
    Lu, Z.J. and Mathews, D. H. (2008) Efficient siRNA selection using hybridization thermodynamics. Nucleic Acids Res. 36, 640-647CrossRefPubMedGoogle Scholar
  39. 39.
    Tafer, H., Ameres, S. L., Obernosterer, G., Gebeshuber, C.A., Schroeder, R., Martinez, J. and Hofacker, I.L. (2008) The impact of target site accessibility on the design of effective siRNAs. Nat. Biotechnol. 26, 578–583.CrossRefPubMedGoogle Scholar
  40. 40.
    Boese, Q., Leake, D., Reynolds, A., Read, S., Scaringe, S.A., Marshall, W. S .and Khvorova, A. (2005) Mechanistic insights aid computational short interfering RNA design. Methods Enzymol. 392, 73–96.CrossRefPubMedGoogle Scholar
  41. 41.
    Mückstein, U., Tafer, H., Hackermüller, J., Bernhart, S. H., Stadler, P. F. and Hofacker, I. L. (2006) Thermodynamics of RNA-RNA binding. Bioinformatics 22, 1177–1182.CrossRefPubMedGoogle Scholar
  42. 42.
    Mückstein, U., Tafer, H., Bernhart, S. H., Hernandez-Rosales, M., Vogel, J., Stadler, P. F. and Hofacker, I. L. (2008) Translational control by RNA-RNA interaction: Improved computation of RNA-RNA binding thermodynamics, In: Bioinformatics research and development (vol. 13), Communications in computer and information science (Elloumi, M., Kung. J., Linial, M., Murphy, R., Schneider, K., and Toma, C., eds.). Springer, pp. 114–127.Google Scholar
  43. 43.
    Hornung, V., Guenthner-Biller, M., Bourquin, C., a Ablasser, A., Schlee, M., Uematsu, S. et al. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263–270.CrossRefPubMedGoogle Scholar
  44. 44.
    de Haro, C., Méndez, R. and Santoyo, J. (1996) The eIF-2alpha kinases and the control of protein synthesis. FASEB J. 10, 1378–1387.PubMedGoogle Scholar
  45. 45.
    Marques, J.T, and Williams, B. R. G. (2005) Activation of the mammalian immune system by siRNAs. Nat. Biotechnol. 23, 1399–1405.CrossRefPubMedGoogle Scholar
  46. 46.
    Shao, X-D., Wu, K-C., Guo, X-Z., Xie, M-J., Zhang, J. and Fan, D-M. (2008) Expression and significance of HERG protein in gastric cancer. Cancer Biol. Ther. 7, 45–50.CrossRefPubMedGoogle Scholar
  47. 47.
    Ding, Y. and Lawrence, C. E. (2003) A statistical sampling algorithm for RNA secondary structure prediction. Nucleic Acids Res. 31, 7280–7301.CrossRefPubMedGoogle Scholar
  48. 48.
    Harborth, J., Elbashir, S.M., Vandenburgh, K., Manninga, H., Scaringe, S.A., Weber, K. and Tuschl, T. (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev. 13, 83–105.CrossRefPubMedGoogle Scholar
  49. 49.
    Ladunga, I. (2007) More complete gene silencing by fewer siRNAs: transparent optimized design and biophysical signature. Nucleic Acids Res. 35, 433–440.CrossRefPubMedGoogle Scholar
  50. 50.
    Bernhart, S.H., Tafer, H., Mückstein, U., Flamm, C., Stadler, P.F. and Hofacker , I.L. (2006) Partition function and base pairing probabilities of RNA heterodimers. Algorithms Mol. Biol. 1, 3CrossRefPubMedGoogle Scholar
  51. 51.
    Bompfünewerer, A. F., Backofen, R., Bernhart, S.H., Hertel, J., Hofacker, I. L., Stadler, P. F. and Will, S. (2008) Variations on RNA folding and alignment: Lessons from Benasque. J. Math. Biol. 56, 119–144.Google Scholar
  52. 52.
    Haley, B., and Zamore, P.D. (2004) Kinetic analysis of the RNAi enzyme complex. Nat. Struct. Mol. Biol. 11, 599–606.CrossRefPubMedGoogle Scholar
  53. 53.
    Jackson, A.L., Bartz, S.R., Schelter, J., Kobayashi, S.V., Burchard, J., Mao, M., et al. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Institute for Theoretical ChemistryUniversity ViennaViennaAustria

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