Kim, D.H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222-226

Nature Biotechnology (Impact Factor: 41.51). 12/2004; 23(2):222-226. DOI: 10.1038/nbt1051
Source: PubMed


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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Available from: John J Rossi, Jun 22, 2015
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    • "Moreover, it has been demonstrated that the synthetic 19 nts siRNAs mediate gene inhibition efficiently in the cytoplasm in vitro [1]–[5] or in vivo [6]. However, it has been reported that the synthetic 25–30 nts RNA duplexes were more potent than their 19 nts long counterparts, especially the 21-mer siRNAs with no overhang [26]. Zamore and colleagues noted that siRNA duplexes were functionally asymmetric [27]. "
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    ABSTRACT: Small interfering RNAs (siRNAs) are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs) could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a) complementary to the TATA-box-centered region; (b) UA usage at the first two bases of the antisense strand; (c) twenty-three nucleotides (nts) in length; (d) 2'-O-Methyl (2'-OMe) modification at the 3' terminus of the antisense strand; (e) avoiding mismatches at the 3' end of the antisense strand. The optimized activating siRNAs potently enhance the expression of interleukin-2 (IL-2) gene in human and mouse primary CD4+ T cells with a long-time effect. Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "With large numbers of transcripts generated from the U6 promoter, these transcripts have a high probability of forming dimers, which are much more stable (having lower ΔG). The dimers can be processed by Dicer into duplexes22,23,24,25 that can be loaded into Ago1, 3, or 4. We hypothesized that stabilizing the stem of native pre-miR-451 might reduce the propensity of forming dimers and therefore, improve the Ago2 specificity. We changed the nucleotides at positions 23, 35, and 40 to make a miR-451 with a completely complementary stem (miR-451-m0, Figure 2b). "
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    ABSTRACT: Relatively large amounts of transfected siRNA can compete for Ago proteins and thus compromise endogenous miRNA function, potentially leading to toxicities. Here, we show that shRNA can also perturb endogenous miRNA function similarly. More importantly, we also show that the problem can be solved by designing shRNAs in the context of pre-miR-451 structure with completely complementary stem, which significantly improves the Ago2 specificity. This shRNA was shown to be Ago2-specific, and maintain target-silencing ability while avoiding competition with endogenous miRNAs by not competing for Agos 1, 3, and 4. We conclude that modified pre-miR-451 structure provides a general platform to design shRNAs that significantly reduce perturbation of miRNA function.
    Full-text · Article · Jul 2014 · Molecular Therapy - Nucleic Acids
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    • "are available to predict effective siRNA based on common features with limitations over in vitro approach ( Pei & Tuschl , 2006 ; Reynolds et al . , 2004 ) . Reports have shown that incorporation of slightly longer siRNA found to have better silencing but longer siRNA is prone to activation of immune response and often more complicated to design ( Kim et al . , 2005 ; Reynolds et al . , 2006 ) . It is possible to manipulate strand for better selection by making a single nucleotide substitution at the end of the duplex to alter the relative binding of the ends ( Schwarz et al . , 2003 ) . By designing a siRNA with a mismatch at the 5′ end of the intended active strand that favors its incorporation i"
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    ABSTRACT: Long-term research on RNA interference led to an unfathomed understanding of the mechanism of siRNA-mediated silencing and finally siRNA has emerged as a powerful therapeutic tool. With siRNAs virtually every gene in the human genome contributing to a disease becomes amenable to regulation, thus opening unprecedented opportunities for drug discovery. siRNA has a well-established role as a tool for in vitro target screening and validation, besides these recent progresses of siRNA delivery in vivo, this has raised more expectations for siRNA-based drugs as the up-and-coming 'magic bullet'. Although a plethora of articles have been published with siRNA, the fundamentals of siRNA-mediated gene silencing and transforming the functional genomics to novel therapeutics are reviewed in this article with consideration to present hurdles as a new generation challenge.
    Full-text · Article · Jul 2014 · Biotechnology & genetic engineering reviews
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