Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Synthetic shRNAs as potent RNAi triggers


Designing potent silencing triggers is key to the successful application of RNA interference (RNAi) in mammals. Recent studies suggest that the assembly of RNAi effector complexes is coupled to Dicer cleavage. Here we examine whether transfection of optimized Dicer substrates results in an improved RNAi response. Dicer cleavage of chemically synthesized short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3′ overhangs produced predictable homogeneous small RNAs comprising the 22 bases at the 3′ end of the stem. Consequently, direct comparisons of synthetic small interfering RNAs and shRNAs that yield the same small RNA became possible. We found synthetic 29-mer shRNAs to be more potent inducers of RNAi than small interfering RNAs. Maximal inhibition of target genes was achieved at lower concentrations and silencing at 24 h was often greater. These studies provide the basis for an improved approach to triggering experimental silencing via the RNAi pathway.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: In vitro processing of 29-bp shRNAs by Dicer generates a predominant siRNA from the end of each short hairpin.
Figure 2: Primer extension analysis shows that similar small RNAs are generated by Dicer processing in vitro or in vivo.
Figure 3: Gene suppression by shRNAs is comparable to or more effective than that achieved by siRNAs targeting the same sequences.
Figure 4: Microarray profiling shows that gene expression profiles of 29-mer shRNAs and the corresponding siRNAs are more similar than expression profiles of 19-mer shRNAs and the corresponding siRNAs.


  1. 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).

    CAS  Article  Google Scholar 

  2. Hammond, S.M., Bernstein, E., Beach, D. & Hannon, G.J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000).

    CAS  Article  Google Scholar 

  3. 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).

    CAS  Article  Google Scholar 

  4. Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R. & Hannon, G.J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 (2001).

    CAS  Article  Google Scholar 

  5. Song, J.J., Smith, S.K., Hannon, G.J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).

    CAS  Article  Google Scholar 

  6. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  8. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    CAS  Article  Google Scholar 

  9. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  11. Ketting, R.F. et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15, 2654–2659 (2001).

    CAS  Article  Google Scholar 

  12. Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34 (2001).

    CAS  Article  Google Scholar 

  13. Brummelkamp, T.R., Bernards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002).

    CAS  Article  Google Scholar 

  14. Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J. & Conklin, D.S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002).

    CAS  Article  Google Scholar 

  15. Zeng, Y., Wagner, E.J. & Cullen, B.R. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333 (2002).

    CAS  Article  Google Scholar 

  16. Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    CAS  Article  Google Scholar 

  17. Khvorova, A., Reynolds, A. & Jayasena, S.D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    CAS  Article  Google Scholar 

  18. Lee, Y.S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004).

    CAS  Article  Google Scholar 

  19. Pham, J.W., Pellino, J.L., Lee, Y.S., Carthew, R.W. & Sontheimer, E.J. A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117, 83–94 (2004).

    CAS  Article  Google Scholar 

  20. Tomari, Y. et al. RISC assembly defects in the Drosophila RNAi mutant armitage. Cell 116, 831–841 (2004).

    CAS  Article  Google Scholar 

  21. Zhang, H., Kolb, F.A., Brondani, V., Billy, E. & Filipowicz, W. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885 (2002).

    CAS  Article  Google Scholar 

  22. Lund, E., Guttinger, S., Calado, A., Dahlberg, J.E. & Kutay, U. Nuclear export of microRNA precursors. Science 303, 95–98 (2004).

    CAS  Article  Google Scholar 

  23. Ma, J.B., Ye, K. & Patel, D.J. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318–322 (2004).

    CAS  Article  Google Scholar 

  24. Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain. Nature 426, 465–469 (2003).

    CAS  Article  Google Scholar 

  25. Song, J.J. et al. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nat. Struct. Biol. 10, 1026–1032 (2003).

    CAS  Article  Google Scholar 

  26. Yan, K.S. et al. Structure and conserved RNA binding of the PAZ domain. Nature 426, 468–474 (2003).

    Article  Google Scholar 

  27. Zhang, H., Kolb, F.A., Jaskiewicz, L., Westhof, E. & Filipowicz, W. Single processing center models for human Dicer and bacterial RNase III. Cell 118, 57–68 (2004).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  29. Rossi, J.J. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, in the press (2005).

  30. Doi, N. et al. Short-interfering-RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors. Curr. Biol. 13, 41–46 (2003).

    CAS  Article  Google Scholar 

Download references


G.J.H. is supported by an Innovator Award from the U.S. Army Breast Cancer Research Program. This work was also supported by a grant from the US National Institutes of Health (G.J.H.). D.S. is supported by a predoctoral fellowship from the US Army Breast Cancer Research Program. We thank the Rosetta Gene Expression Laboratory for microarray RNA processing and hybridizations.

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Gregory J Hannon or Michele A Cleary.

Ethics declarations

Competing interests

A number of the authors of this paper are employed by a for-profit company, Rosetta Inpharmatics, a wholly owned subsidiary of Merck. Partial funding for the work described in this paper was provided by Merck.

Supplementary information

Supplementary Fig. 1

The set of shRNAs containing 19 or 29 bp. stems coding a luciferase sequence and either bearing or lacking a 2 nt. 3′ overhang were incubated with bacterial RNase III to verify their double-stranded nature. (PDF 103 kb)

Supplementary Fig. 2

Northern blotting indicates that siRNAs and 19mer and 29mer shRNAs all give rise to RISC. (PDF 64 kb)

Supplementary Fig. 3

Structures of synthetic RNAs used for comparing siRNA and shRNA. (PDF 139 kb)

Supplementary Table 1

Sequences of the siRNAs used in this study. (PDF 35 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Siolas, D., Lerner, C., Burchard, J. et al. Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol 23, 227–231 (2005).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing