A fluorescence-based assay for multisubunit DNA-dependent RNA polymerases
A fluorescence-based assay for multisubunit DNA-dependent RNA polymerases
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DOI：
10.1016/j.ab.2003.08.038
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发表时间：
2004-01-15
影响因子：
2.9
通讯作者：
Galant, A
中科院分区：
文献类型：
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作者：
Kuhlman, P;Duff, HL;Galant, A
自20世纪60年代以来，DNA依赖性RNA聚合酶的性质已经被研究，但在探测RNA聚合酶结构/功能关系，不同类别的RNA聚合酶在细胞过程中的作用以及使用RNA聚合酶作为药物靶点的可行性方面仍然存在相当大的兴趣。历史上，RNA聚合酶活性通过将放射性同位素标记的核苷酸掺入RNA中来测量。我们报告了一种RNA聚合酶活性测定方法的发展，该方法使用染料RiboGreen通过荧光检测转录本，因此不需要放射性同位素的费用、保质期短和处理成本高。该方法相对快速，可以完全以微孔板形式进行，允许并行处理数十至数百个样品。因此，它应该是非常适合用于药物筛选和分析色谱馏分。由于RiboGreen荧光通过与RNA或DNA结合而增强，因此必须在测定程序的转录和检测阶段之间通过DNA酶消化和超滤去除模板DNA。虽然RiboGreen荧光对溶剂环境的变化敏感，但超滤步骤中的溶剂交换允许即使在盐、pH等极端条件下也能比较转录水平。All rights reserved.
The properties of DNA-dependent RNA polymerases have been studied since the 1960s, but considerable interest in probing RNA polymerase structure/function relationships, the roles of different classes of RNA polymerases in cellular processes, and the feasibility of using RNA polymerases as drug targets still exists. Historically, RNA polymerase activity has been measured by the incorporation into RNA of radioisotopically labeled nucleotides. We report the development of an assay for RNA polymerase activity that uses the dye RiboGreen to detect transcripts by fluorescence and is thus free of the expense, short shelf life, and high handling costs of radioisotopes. The method is relatively quick and can be performed entirely in microplate format, allowing for the processing of dozens to hundreds of samples in parallel. It should thus be well-suited to use in drug screening and analysis of chromatographic fractions. As RiboGreen fluorescence is enhanced by binding to either RNA or DNA, template DNA must be removed by DNase digestion and ultrafiltration between the transcription and the detection phases of the assay procedure. Although RiboGreen fluorescence is sensitive to changes in solvent environment, solvent exchange in the ultrafiltration step allows comparison of transcription levels even under extremes of salt, pH, etc. (C) 2003 Elsevier Inc. All rights reserved.