Amarzguioui, M. et al. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat. Protocols 1, 508-517

The Biotechnology Centre of Oslo, Gaustadalleen 21, 0349 Oslo, Norway.
Nature Protocols (Impact Factor: 9.67). 06/2006; 1(2):508-517. DOI: 10.1038/nprot.2006.72
Source: PubMed


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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    • ".com/?barid¼{327C53A4-FC6B-11E2-AB6C-0030679F4C46} &src¼97&crg¼3.5000006.10042&st¼23. Accessed January 14, 2015.) is a gain of function or toxic effect of the mutant protein, other strategies, such as small-interfering RNA to silence the abnormal copy of the gene, must be used [8] [9].They have been studied in neurodegenerative disorders [10]. A new technology , Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR), which uses a Cas9 enzyme, may offer an alternative to gene replacement therapy [11] [12]. "

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    • "The predicted masses for the oligonucleotides were verified by electrospray-ionization mass spectrometry (ESI-MS) and were within ±0.02%. The protocol for DsiRNA design and manufacture has been described in detail.45,46 The DIG-labeled DsiRNA was made by attaching DIG-NHS ester an internal-dT residue in a 2-O′ methyl modified pig-HPRT-specific DsiRNA. "
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    ABSTRACT: Well-differentiated human airway epithelia present formidable barriers to efficient siRNA delivery. We previously reported that treatment of airway epithelia with specific small molecules improves oligonucleotide uptake and facilitates RNAi responses. Here, we exploited the platelet activating factor receptor (PAFR) pathway, utilized by specific bacteria to transcytose into epithelia, as a trigger for internalization of Dicer-substrate siRNAs (DsiRNA). PAFR is a G-protein coupled receptor which can be engaged and activated by phosphorylcholine residues on the lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae and the teichoic acid of Streptococcus pneumoniae as well as by its natural ligand, platelet activating factor (PAF). When well-differentiated airway epithelia were simultaneously treated with either nontypeable Haemophilus influenzae LOS or PAF and transduced with DsiRNA formulated with the peptide transductin, we observed silencing of both endogenous and exogenous targets. PAF receptor antagonists prevented LOS or PAF-assisted DsiRNA silencing, demonstrating that ligand engagement of PAFR is essential for this process. Additionally, PAF-assisted DsiRNA transfection decreased CFTR protein expression and function and reduced exogenous viral protein levels and titer in human airway epithelia. Treatment with spiperone, a small molecule identified using the Connectivity map database to correlate gene expression changes in response to drug treatment with those associated with PAFR stimulation, also induced silencing. These results suggest that the signaling pathway activated by PAFR binding can be manipulated to facilitate siRNA entry and function in difficult to transfect well-differentiated airway epithelial cells.
    Full-text · Article · Jul 2014 · Molecular Therapy - Nucleic Acids
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    • "We believe that PHDcleav have the potential to be used in future investigations on genetic variations in miRNA loci and their effect on speed and accuracy of Dicer processing, and its impact on target gene silencing. Furthermore, studies have shown that use of Dicer specific siRNA can improve the RNAi silencing [65,66]. This tool will also be useful in the design and careful selection of shRNA/amiRNA for more potent gene silencing. "
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    ABSTRACT: Background: Dicer, an RNase III enzyme, plays a vital role in the processing of pre-miRNAs for generating the miRNAs. The structural and sequence features on pre-miRNA which can facilitate position and efficiency of cleavage are not well known. A precise cleavage by Dicer is crucial because an inaccurate processing can produce miRNA with different seed regions which can alter the repertoire of target genes. Results: In this study, a novel method has been developed to predict Dicer cleavage sites on pre-miRNAs using Support Vector Machine. We used the dataset of experimentally validated human miRNA hairpins from miRBase, and extracted fourteen nucleotides around Dicer cleavage sites. We developed number of models using various types of features and achieved maximum accuracy of 66% using binary profile of nucleotide sequence taken from 5p arm of hairpin. The prediction performance of Dicer cleavage site improved significantly from 66% to 86% when we integrated secondary structure information. This indicates that secondary structure plays an important role in the selection of cleavage site. All models were trained and tested on 555 experimentally validated cleavage sites and evaluated using 5-fold cross validation technique. In addition, the performance was also evaluated on an independent testing dataset that achieved an accuracy of ~82%. Conclusion: Based on this study, we developed a webserver PHDcleav ( to predict Dicer cleavage sites in pre-miRNA. This tool can be used to investigate functional consequences of genetic variations/SNPs in miRNA on Dicer cleavage site, and gene silencing. Moreover, it would also be useful in the discovery of miRNAs in human genome and design of Dicer specific pre-miRNAs for potent gene silencing.
    Full-text · Article · Oct 2013 · BMC Bioinformatics
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