Pfizer Research Technology Center, Cambridge, MA, USA.
Biotechniques. 2010 Apr;48(4):303-11. doi: 10.2144/000113402.
The cleavage of double-stranded RNA (dsRNA) molecules by Dicer is a critical step in silencing genes by the RNA induced silencing complex (RISC). The development of Dicer substrates as nucleic acid-based therapeutics brings about a need to rapidly evaluate chemically modified RNA molecules for their ability to be processed by Dicer. This study outlines a quantitative fluorescence quencher-based assay for studying the ability of Dicer substrates to be processed by Dicer. By using a dsRNA probe labeled with Cy5-Iowa Black RQ, a panel of unlabeled test substrates can be rapidly assessed in heterologous competition assays without the need for electrophoresis or radiolabeling. This assay was piloted by evaluation of 196 unlabeled 27-mer Dicer substrates with various overhang structures in a purified Dicer enzyme assay system. Results indicate that Dicer has no preference for the sequence of RNA in the main double-stranded region of the substrate. However, a preference for Dicer substrate RNAs (D-siRNAs) containing purine/purine 3' overhang nucleotides was observed. These results demonstrate that the method is useful for studying the effects of modified nucleic acids in addition to rapidly accessing the influence of potential regulatory factors on Dicer processing.
双链 RNA (dsRNA) 分子的切割是 RNA 诱导沉默复合物 (RISC) 沉默基因的关键步骤。Dicer 底物作为核酸治疗药物的发展带来了一个需要快速评估化学修饰 RNA 分子被 Dicer 加工的能力的需求。本研究概述了一种基于定量荧光猝灭剂的测定法，用于研究 Dicer 底物被 Dicer 加工的能力。通过使用 Cy5-Iowa Black RQ 标记的 dsRNA 探针，可以在异源竞争测定中快速评估一组未标记的测试底物，而无需电泳或放射性标记。该测定法通过在纯化的 Dicer 酶测定系统中评估具有各种突出结构的 196 种未标记的 27 个碱基对 Dicer 底物进行了试点研究。结果表明，Dicer 对底物中双链区域内 RNA 的序列没有偏好。然而，观察到含有嘌呤/嘌呤 3'突出核苷酸的 Dicer 底物 RNA（D-siRNA）具有偏好性。这些结果表明，该方法除了快速评估潜在调节因子对 Dicer 加工的影响外，还可用于研究修饰核酸的影响。
