Potential Design Rules and Enzymatic Synthesis of siRNAs

  • Mouldy Sioud
  • Marianne Leirdal
Part of the Methods in Molecular Biology™ book series (MIMB, volume 252)


Small interfering RNAs (siRNAs) have emerged as a powerful technique for sequence-specific gene silencing in a wide variety of organisms. However, the base composition of the siRNA sequence is not the only determinant of efficacy. Intrinsic factors related to mRNA structures are likely to be crucial determinants for siRNA activity. Indeed, placing the recognition site of an active siRNA into a structured mRNA region has abrogated the siRNA activity. Therefore, a successful gene-targeting project may require the design of many distinct siRNAs at a high cost. Here, potential design rules, cost-effective strategies for producing siRNAs by T7 RNA polymerase, and expression cassettes for in vivo testing are described.


Antisense Strand siRNA Duplex Primer Extension Reaction Target Accessibility Effective siRNAs 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Sioud, M. (2001) Nucleic acid enzymes as a novel generation of anti-gene agents. Curr. Mol. Med. 1, 575–588.PubMedCrossRefGoogle Scholar
  2. 2.
    Sharp, P. A. (2001) RNA interference. Genes Dev. 15, 188–200.CrossRefGoogle Scholar
  3. 3.
    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, 806–811.PubMedCrossRefGoogle Scholar
  4. 4.
    Hamilton, A. J. and Baulcombe, D. C. (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952.PubMedCrossRefGoogle Scholar
  5. 5.
    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, 494–498.PubMedCrossRefGoogle Scholar
  6. 6.
    Sorrensen, D. R., Leirdal, M., and Sioud, M. (2003) Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J. Mol. Biol. 327, 761–766CrossRefGoogle Scholar
  7. 7.
    Lewis, D. L., Hagstom, J. E., Loomis, A. G., Wolff, J. A., and Herweijer, H. (2002) Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nature Genet. 32, 107,108.PubMedCrossRefGoogle Scholar
  8. 8.
    McManus, M. T. and Sharp, P. A. (2002) Gene silencing in mammalians by siRNAs. Nat. Genet. Rev. 3, 737–740.CrossRefGoogle Scholar
  9. 9.
    Bennett, C. F., Chiang, M.-Y., Wilson-Lingardo, L., and Wyatt, J. R. (1994) Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides. Nucleic Acids Res. 22, 3202–3209.PubMedCrossRefGoogle Scholar
  10. 10.
    Sioud, M. (1997) Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates. Nucleic Acids Res. 25, 333–338.PubMedCrossRefGoogle Scholar
  11. 11.
    Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M.J., Ehsani, A., et al. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotech. 20, 500–505.Google Scholar
  12. 12.
    Sczakiel, G., Homann, M., and Rittner, K. (1993) Compute-aided search for effective antisense RNA target sequences of the human immunodeficiency virus type 1. Antisense Res. Dev. 3, 45–52.PubMedGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    Leirdal, M. and Sioud, M. (2002). Gene silencing in mammalian cells by preformed small RNA duplexes. Biochem. Biophys. Res. Commun. 295, 744–748.PubMedCrossRefGoogle Scholar
  15. 15.
    Jacque, J. M., Triques, K., and Stevenson, M. (2002) Modulation of HIV-1 replication by RNA interference. Nature 896, −4.Google Scholar
  16. 16.
    Vickers, T. A., Koo, S., Bennet, 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. J. Biol. Chem. 278, 7108–7118.PubMedCrossRefGoogle Scholar
  17. 17.
    Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553.PubMedCrossRefGoogle Scholar
  18. 18.
    Paul, C. P., Green, P. D., Winer, I., and Engelke, D. R. (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 19, 505–508.CrossRefGoogle Scholar
  19. 19.
    Miyagishi, M. and Taira, K. (2002) U6 promoter-driven siRNA with four uridines 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 19, 497–500.CrossRefGoogle Scholar
  20. 20.
    Hannon, G. J. (2002) RNA interference. Nature 418, 244–251.PubMedCrossRefGoogle Scholar
  21. 21.
    Castanotto, D., Li, H., and Rossi, J. J. (2002) Functional siRNA expression from transfected PCR products RNA, 8, 1454–1460.PubMedCrossRefGoogle Scholar
  22. 22.
    Doech, J. G., Petersen, C. P., and Sharp, P. A. (2003) siRNA can function as miRNAs. Genes Devel. 17, 438–442.CrossRefGoogle Scholar
  23. 23.
    Sioud, M. and Jespersen, L. (1996) Enhancement of hammerhead ribozyme catalysis by glyreraldehyde-3-phophate dehydrogenase. J. Mol. Biol. 257, 775–789.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Mouldy Sioud
    • 1
  • Marianne Leirdal
    • 1
  1. 1.Department of Immunology, Molecular Medicine GroupThe Norwegian Radium HospitalOsloNorway

Personalised recommendations