Homogeneous fluorescent assay for RNA polymerase
Homogeneous fluorescent assay for RNA polymerase
复制标题
DOI：
10.1016/j.ab.2005.04.022
复制
发表时间：
2005-07-15
影响因子：
2.9
通讯作者：
Mustaev, A
中科院分区：
文献类型：
--
作者：
Kozlov, M;Bergendahl, V;Mustaev, A
提出了一种测定RNA聚合酶(RNAP)活性的新方法。该方法使用在磷酸盐末端携带4-甲基伞形酮残基的核苷三磷和四磷酸衍生物。通过RNA聚合酶将这些化合物结合到RNA中，伴随着4-甲基伞形酮的二磷酸和三磷酸衍生物的释放。随后用碱性磷酸酶处理产生游离的4-甲基伞形酮，它是高度荧光的，很容易被检测到。该方法的灵敏度比以往研究报道的要高。通过从16,000种化合物中检索RNAP抑制剂，证明了该分析的有效性。(C)2005 Elsevier Inc.保留所有权利。
A new method for determination of RNA polymerase (RNAP) activity is presented. The method uses nucleoside tri- and tetraphosphate derivatives carrying 4-methylumbelliferone residue at the terminal phosphate. Incorporation of such compounds in RNA by RNA polymerase is accompanied by release of di- and triphosphate derivatives of 4-methylumbelliferone. Subsequent treatment by alkaline phosphatase produces free 4-methylumbelliferone that is highly fluorescent and can be easily detected. The sensitivity of the method is higher than that reported in previous studies. The validity of the assay has been demonstrated by retrieving the RNAP inhibitors from a collection of 16,000 compounds. (c) 2005 Elsevier Inc. All rights reserved.