Podolska Katerina, Sedlak David, Bartunek Petr, Svoboda Petr
1Institute of Molecular Genetics of the ASCR, v.v.i., Prague 4, Czech Republic.
J Biomol Screen. 2014 Mar;19(3):417-26. doi: 10.1177/1087057113497400. Epub 2013 Aug 14.
Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.
核糖核酸酶III Dicer产生小RNA是微小RNA和RNA干扰途径中的关键步骤，这些途径利用Dicer产生的小RNA作为序列特异性沉默向导。对微小RNA和RNA干扰途径的进一步研究及操作将受益于小分子调节剂的鉴定。在此，我们报告了一项基于荧光的体外Dicer切割测定法的研究，该方法适用于高通量筛选。动力学测定可在单轮反应条件下（35 nM底物和70 nM Dicer）于小体积（5 µL）中进行，这使其适用于1536孔板形式的高通量筛选。作为原理验证，分析了一个生物活性化合物小文库，证明了该测定法的潜力。
