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Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy

Nature Biotechnology volume 23, pages 222–226 (2005)Cite this article

Abstract

RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNAs1,2,3. In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes 25–30 nucleotides in length can be up to 100-fold more potent than corresponding conventional 21-mer small interfering RNAs (siRNAs). Some sites that are refractory to silencing by 21-mer siRNAs can be effectively targeted by 27-mer duplexes, with silencing lasting up to 10 d. Notably, the 27-mers do not induce interferon or activate protein kinase R (PKR). The enhanced potency of the longer duplexes is attributed to the fact that they are substrates of the Dicer endonuclease, directly linking the production of siRNAs to incorporation in the RNA-induced silencing complex. These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.

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Figure 1: 27-mer dsRNAs are more potent effectors of RNAi than a 21+2 siRNA.
Figure 2: Dicer processing correlates with RNAi activity.
Figure 3: Features of 27-mer dsRNA in RNAi.

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References

  1. Hannon, G.J. RNA interference. Nature 418, 244–251 (2002).

    Article  CAS  Google Scholar 

  2. Hutvagner, G. & Zamore, P.D. RNAi: nature abhors a double-strand. Curr. Opin. Genet. Dev. 12, 225–232 (2002).

    Article  CAS  Google Scholar 

  3. Sharp, P.A. RNAi and double-strand RNA. Genes Dev. 13, 139–141 (1999).

    Article  CAS  Google Scholar 

  4. Napoli, C., Lemieux, C. & Jorgensen, R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279–289 (1990).

    Article  CAS  Google Scholar 

  5. Romano, N. & Macino, G. Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol. Microbiol. 6, 3343–3353 (1992).

    Article  CAS  Google Scholar 

  6. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    Article  CAS  Google Scholar 

  7. Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H. & Schreiber, R.D. How cells respond to interferons. Annu. Rev. Biochem. 67, 227–264 (1998).

    Article  CAS  Google Scholar 

  8. Minks, M.A., Benvin, S., Maroney, P.A. & Baglioni, C. Synthesis of 2′–5′-oligo(A) in extracts of interferon-treated HeLa cells. J. Biol. Chem. 254, 5058–5064 (1979).

    CAS  PubMed  Google Scholar 

  9. Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).

    Article  CAS  Google Scholar 

  10. Zamore, P.D., Tuschl, T., Sharp, P.A. & Bartel, D.P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 (2000).

    Article  CAS  Google Scholar 

  11. Bernstein, E., Caudy, A.A., Hammond, S.M. & Hannon, G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).

    Article  CAS  Google Scholar 

  12. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  13. Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W. & Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888 (2001).

    Article  CAS  Google Scholar 

  14. Kim, D.H. et al. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat. Biotechnol. 22, 321–325 (2004).

    Article  CAS  Google Scholar 

  15. Kim, D.H. & Rossi, J.J. Coupling of RNAi-mediated target downregulation with gene replacement. Antisense Nucleic Acid Drug Dev. 13, 151–155 (2003).

    Article  CAS  Google Scholar 

  16. Persengiev, S.P., Zhu, X. & Green, M.R. Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 12–18 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Scherer, L. & Rossi, J.J. RNAi applications in mammalian cells. Biotechniques 36, 557–561 (2004).

    Article  CAS  Google Scholar 

  19. Ui-Tei, K. et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 32, 936–948 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Markovtsov, V. et al. Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein. Mol. Cell. Biol. 20, 7463–7479 (2000).

    Article  CAS  Google Scholar 

  22. Wolin, S.L. & Cedervall, T. The La protein. Annu. Rev. Biochem. 71, 375–403 (2002).

    Article  CAS  Google Scholar 

  23. Manche, L., Green, S.R., Schmedt, C. & Mathews, M.B. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol. Cell. Biol. 12, 5238–5248 (1992).

    Article  CAS  Google Scholar 

  24. Gunnery, S. & Mathews, M.B. RNA binding and modulation of PKR activity. Methods 15, 189–198 (1998).

    Article  CAS  Google Scholar 

  25. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    Article  CAS  Google Scholar 

  26. Bohula, E.A. et al. 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 (2003).

    Article  CAS  Google Scholar 

  27. Caplen, N.J., Parrish, S., Imani, F., Fire, A. & Morgan, R.A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98, 9742–9747 (2001).

    Article  CAS  Google Scholar 

  28. Siolas, D. et al. Synthetic shRNAs as potent RNAi triggers. Nat. Biotechnol. 23, in the press (2005).

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Acknowledgements

D.K. is a Beckman Fellow. This work was supported by a grant from the Arnold and Mabel Beckman Foundation and the US National Institutes of Health (AI29329 and AI42552, and HL074704 to J.J.R.). The authors wish to dedicate this work to the memory of Arnold Beckman, who recently passed away.

Author information

Authors and Affiliations

  1. Division of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Rd., Duarte, 91010, California, USA

    Dong-Ho Kim & John J Rossi

  2. Integrated DNA Technologies, 1710 Commercial Park, Coralville, 52241, Iowa, USA

    Mark A Behlke & Scott D Rose

  3. Division of Biology, 147-75, California Institute of Technology, Pasadena, 91125, California, USA

    Mi-Sook Chang & Sangdun Choi

  4. Graduate School of Biological Sciences, City of Hope and Beckman Research Institute of the City of Hope, 1450 East Duarte Rd., Duarte, 91010, California, USA

    John J Rossi

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  1. Dong-Ho Kim
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  2. Mark A Behlke
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  4. Mi-Sook Chang
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  5. Sangdun Choi
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  6. John J Rossi
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Corresponding author

Correspondence to John J Rossi.

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Competing interests

M.A.B. and S.D.R. are employed by Integrated DNA Technologies, an institution that may gain or lose financially as a result of the publication of this article.

Supplementary information

Supplementary Fig. 1 (PDF 230 kb)

Supplementary Fig. 2

ESI mass spectra of the 27mer duplex EGFPS1 27+0 before (top) and after (bottom) incubation with Dicer are shown. (PDF 125 kb)

Supplementary Fig. 3

Sequence specificity of Dicer substrate 27mer dsRNAs. (PDF 156 kb)

Supplementary Fig. 4

SiRNAs and Dicer substrate dsRNAs do not induce interferons or activate PKR or generate specific “off target effects”. (PDF 253 kb)

Supplementary Table 1

Summary of oligonucleotide reagents. (PDF 113 kb)

Supplementary Table 2

Molecular weights of possible 21mer duplexes derived from the 27mer duplex by Dicer processing. (PDF 107 kb)

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Kim, DH., Behlke, M., Rose, S. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol 23, 222–226 (2005). https://doi.org/10.1038/nbt1051

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