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RNA interference (RNAi) begins when Dicer recognizes a dsRNA precursor and cleaves it into a 21–23-bp short interfering RNA (siRNA) that usually contains a 2-nucleotide overhang at the 3′ end. This resulting siRNA then combines with the RNA-induced silencing complex (RISC), leading to degradation of the corresponding mRNA through the endonuclease activity of the argonaute protein. Genes can be silenced by delivering siRNAs directly to the cell, but studies have shown that Dicer-substrate RNAs (D-siRNAs) elicit more efficient mRNA cleavage, presumably through Dicer interaction with cofactors in the RNAi pathway. Increased interest in delivering Dicer substrates for therapeutic purposes has spurred development of assays to screen for potential D-siRNAs, but to date, these methods have been complicated by their reliance on time-consuming RNA radiolabeling and native or denaturing gel electrophoresis. In an effort to identify a rapid, high-throughput approach to D-siRNA screening, J. DiNitto and colleagues at Pfizer Research Technology Center (Cambridge, MA) created a heterologous competition assay using a fluorescently labeled Dicer probe substrate, which they describe in this issue. The probe consists of a 29-mer dsRNA Dicer substrate labeled with Cy5 at the 5′ end of the antisense strand and Iowa Black RQ quencher at the 3′ end of the sense strand. Dicer cleavage releases a 5–7-nucleotide oligomer carrying the fluorophore and quencher. This oligomer is rapidly denatured, releasing the quencher strand and leading to a linear increase in fluorescence intensity proportional to Dicer concentration and activity. To test their assay, the authors studied the impact of 3′ overhangs on Dicer activity using four fluorescent Dicer substrate probes in competition with 196 unlabeled D-siRNAs of varying structure. They found that Dicer showed no preference for sense or antisense 2-nucleotide overhangs, but preferred substrates with purine/purine overhangs over pyrimidine/pyrimidine overhangs. This new competition assay presents a rapid method for identifying Dicer substrates for in vivo confirmation and provides a means to evaluate modified substrates and regulators of Dicer activity such as protein co-factors or inhibitors.
SpinelessSite-specific recombination mediated by Cre and FLP recombinases permits the insertion of a transgenic construct into specific docking sites (i.e., recombinase recognition sequences) engineered in the genome. Although this is an important tool for transgenesis of eukaryotic cells, a bacterial backbone (BB) is inserted along with the transgene from the construct plasmid in most protocols, which may negatively affect transgene expression as well as introduce undesired bacterial DNA sequences containing antibiotic resistance genes. While the alternative method of recombination-mediated cassette exchange does exclude the BB from insertion, it requires tandem incompatible recombination sites, precluding the use of many common transgenesis vectors, cell lines, and animal lines developed for insertion at a single docking site. J.E. Jakobsen and colleagues at the University of Aarhus (Aarhus, Denmark) propose a simple but efficient alternative technique for excluding the BB prior to site-specific insertion of a transgene at a single docking site. By PCR-amplifying the transgene cassette from a plasmid construct and then self-ligating the resulting fragments into minicircles, donor DNA lacking a BB can be generated for site-directed transgenesis at a single integration site. They validated their method using a system comprised of FLP recombinase, a donor transgene plasmid containing an RFP gene (DsRed) and the FRT recognition site for FLP-mediated recombination, and cell lines with FRT docking sites. Minicircle donor DNA generated nearly the same number of FLP-dependent transgenic colonies as its parental plasmid construct, and the minicircles were precisely recombined into the docking sites. The resulting integrants lacked the BB, clearly demonstrating that minicircles lacking BB are suitable donor DNA. Similar numbers of colonies expressed DsRed with equally homogeneous expression within the colonies. When the expression level of DsRed was examined more closely by quantitative reverse-transcription PCR, colonies derived from plasmid donor DNA showed 1.5–2 × lower expression compared to minicirclederived colonies, demonstrating that the BB can have a negative effect on transgene expression in some situations. Thus, the use of minicircles as donor DNA may be preferable to plasmid DNA for recombination at a single docking site, as it can reduce potentially negative effects of BBs on transgene expression levels.
