Synthetic shRNAs as potent RNAi triggers

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Abstract

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.

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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.

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Acknowledgements

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

Correspondence to Gregory J Hannon or Michele A Cleary.

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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)

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Siolas, D., Lerner, C., Burchard, J. et al. Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol 23, 227–231 (2005) doi:10.1038/nbt1052

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