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Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy

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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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.

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Correspondence to John J Rossi.

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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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