Homogeneous fluorescent assay for RNA polymerase

Maxim Kozlov, Veit Bergendahl, Richard Burgess, Alex Goldfarb, Arkady Mustaev

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)206-213
Number of pages8
JournalAnalytical Biochemistry
Volume342
Issue number2
DOIs
StatePublished - Jul 15 2005
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Keywords

  • 4-Methylumbelliferone
  • Fluorescent assay
  • High-throughput screening
  • In vitro transcription
  • RNA polymerase

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