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Designing and Utilization of siRNAs Targeting RNA Binding Proteins

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 488)

Summary

Small interfering RNA (siRNA)-mediated RNA interference (RNAi) is a very powerful tool for triggering posttranscriptional gene silencing in several organisms. We discuss the improvement of two different sources of siRNAs synthesized either chemically or by an enzymatic method. When the siRNAs are synthesized by in vitro transcription using a phage polymerase, the initiating triphosphates trigger a potent interferon induction that can lead to misinterpretation of the data. A novel method is presented to minimize the nonspecific effect of enzymatic siRNAs while maintaining the advantages of lower cost and less turnaround time. When chemical siRNAs are used, the expense and long turnaround time can be a problem, especially if the selected siR-NAs are not highly functional in triggering RNAi. The new format for making double-stranded RNAs (dsRNAs) is described to achieve more efficient suppression. The format has been tested by creating siRNAs targeting two RNA binding proteins, La and hnRNP (heterogeneous nuclear ribonucleoprotein) H, and has shown better potency at lower concentrations than the conventional 21-mer siRNA.

Key Words

Dicer Dicer substrates in vitro transcription RNA RNA interference siRNA triphosphate 
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References

  1. 1.
    Hannon, G. J. (2002) RNA interference. Nature 418, 244–251.CrossRefPubMedGoogle 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, 494–498.CrossRefPubMedGoogle Scholar
  3. 3.
    Scherer, L. J., and Rossi, J. J. (2003) Approaches for the sequence-specific knockdown of mRNA. Nat. Biotechnol. 21, 1457–1465.CrossRefPubMedGoogle Scholar
  4. 4.
    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
  5. 5.
    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
  6. 6.
    Sohail, M., Doran, G., Riedemann, J., Macaulay, V. , and Southern, E. M. (2003) A simple and cost-effective method for producing small interfering RNAs with high efficacy. Nucleic Acids Res. 31, e38.CrossRefPubMedGoogle Scholar
  7. 7.
    Donze, O., and Picard, D. (2002) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res. 30, e46.CrossRefPubMedGoogle Scholar
  8. 8.
    Kim, D. H., Longo, M., Han, Y. , Lundberg, P., Cantin, E., and Rossi, J. J. (2004) Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat. Biotechnol. 22, 321–325.CrossRefPubMedGoogle Scholar
  9. 9.
    Zhang, H., Kolb, F. A., Brondani, V. , Billy, E., and Filipowicz, W. (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for AT P. EMBO J. 21, 5875–5885.CrossRefPubMedGoogle Scholar
  10. 10.
    Manche, L., Green, S. R., Schmedt, C., and Mathews, M. B. (1992) Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol. Cell Biol. 12, 5238–5248.PubMedGoogle Scholar
  11. 11.
    Jackson, A. L., Bartz, S. R., Schelter, J., et al. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637.CrossRefPubMedGoogle Scholar
  12. 12.
    Kim, D. H., Behlke, M. A., Rose, S. D., Chang, M. S., Choi, S., and Rossi, J. J. (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226.CrossRefPubMedGoogle Scholar
  13. 13.
    Kim, D. H., and Rossi, J. J. (2003) Coupling of RNAi-mediated target downregu-lation with gene replacement. Antisense Nucleic Acid Drug Dev. 13, 151–155.CrossRefPubMedGoogle Scholar
  14. 14.
    Scherer, L., and Rossi, J. J. (2004) RNAi applications in mammalian cells. Biotechniques 36, 557–561.PubMedGoogle Scholar
  15. 15.
    Wolin, S. L., and Cedervall, T. (2002) The La protein. Annu. Rev. Biochem. 71, 375–403.CrossRefPubMedGoogle Scholar
  16. 16.
    Stefano, J. E. (1984) Purified lupus antigen La recognizes an oligouridylate stretch common to the 3′ termini of RNA polymerase III transcripts. Cell 36, 145–154.CrossRefPubMedGoogle Scholar
  17. 17.
    Markovtsov, V., Nikolic, J. M., Goldman, J. A., Turck, C. W., Chou, M. Y., and Black, D. L. (2000) Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein. Mol. Cell Biol. 20, 7463–7479.CrossRefPubMedGoogle Scholar
  18. 18.
    Chen, C. D., Kobayashi, R., and Helfman, D. M. (1999) Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene. Genes Dev. 13, 593–606.CrossRefPubMedGoogle Scholar
  19. 19.
    Chou, M. Y., Rooke, N., Turck, C. W., and Black, D. L. (1999) hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells. Mol. Cell Biol. 19, 69–77.PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  1. 1.Molecular Biology DivisionBeckman Research Institute of the City of HopeDuarteUSA
  2. 2.Integrated DNA Technologies (IDT)CoralvilleUSA
  3. 3.Molecular Biology DivisionBeckman Research Institute of the City of HopeDuarteUSA

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