A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

• Published in: Analytical biochemistry 2007-06, Vol.365 (2), p.194-200
• Main Authors: Mestas, Santano P., Sholders, Aaron J., Peersen, Olve B.
• Format: Article
• Language: English
• Subjects: Activity assay; Base Sequence; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Fluorescein; Fluorescence polarization; Fluorescence Polarization - methods; Fluorescent Dyes; High-throughput screening; Poliovirus; Polymerase; Promoter Regions, Genetic; RNA - chemistry; RNA - metabolism
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.03.039

We have devised a simple high-throughput screening compatible fluorescence polarization-based assay that can be used to detect the elongation activity of nucleic acid polymerase enzymes. The assay uses a 5′ end-labeled template strand and relies on an increase in the polarization signal from the fluorescent label as it is drawn in toward the active site by the action of the enzyme. If the oligonucleotide is sufficiently short, the fluorescence polarization signal can also be used to detect binding prior to elongation activity. We refer to the nucleic acid substrate as a polymerase elongation template element (PETE) and demonstrate the utility of this PETE assay in a microtiter plate format using the RNA-dependent RNA polymerase from poliovirus to extend a self-priming hairpin RNA. The PETE assay provides an efficient method for screening compounds that may inhibit the nucleic acid binding or elongation activities of polymerases.