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Rational design and in vitro and in vivo delivery of Dicer substrate siRNA

Nature Protocols volume 1, pages 508–517 (2006)Cite this article

Abstract

RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21–25 nt with 2-bp 3′ overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.

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Figure 1: Comparative design of conventional 21-mer and R and L forms of asymmetrical D-siRNA.
Figure 2: psiCheck2 dual luciferase-based reporter cotransfections for monitoring sense and antisense D-siRNA strand selection.
Figure 3: Finalized designs of GAPDH R-form D-siRNA (27R) and their respective 21-mers.
Figure 4: Transfection efficiency using TransIT-TKO and siRNA-Cy3.
Figure 5: Comparison of gene knockdown using D-siRNA and conventional 21-nt siRNA against TNF-α in RAW 264.7 cells.

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Acknowledgements

J.J.R. was supported by National Institutes of Health grants AI29329, AI424552 and HL07470. E.C. was supported by National Institutes of Health grant EY013814.

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Authors and Affiliations

  1. The Biotechnology Centre of Oslo, Gaustadalleen 21, Oslo, 0349, Norway

    Mohammed Amarzguioui

  2. Division of Virology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, 91010, California, USA

    Patric Lundberg & Edouard Cantin

  3. Mirus Bio Corporation, 505 S. Rosa Road, Suite 104, Madison, 53719-1267, Wisconsin, USA

    James Hagstrom

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

    Mark A Behlke

  5. Division of Molecular Biology and Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, 91010, California, USA

    John J Rossi

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  1. Mohammed Amarzguioui
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Correspondence to Mohammed Amarzguioui or John J Rossi.

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J.H. is a co-founder and employee of Mirus Bio Corporation.

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Amarzguioui, M., Lundberg, P., Cantin, E. et al. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat Protoc 1, 508–517 (2006). https://doi.org/10.1038/nprot.2006.72

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