Mestas Santano P, Sholders Aaron J, Peersen Olve B
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
Anal Biochem. 2007 Jun 15;365(2):194-200. doi: 10.1016/j.ab.2007.03.039. Epub 2007 Apr 2.
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.
我们设计了一种简单的、与高通量筛选兼容的基于荧光偏振的检测方法，可用于检测核酸聚合酶的延伸活性。该检测方法使用5'端标记的模板链，并依赖于荧光标记的偏振信号增加，因为它在酶的作用下被拉向活性位点。如果寡核苷酸足够短，荧光偏振信号也可用于在延伸活性之前检测结合。我们将核酸底物称为聚合酶延伸模板元件（PETE），并使用脊髓灰质炎病毒的RNA依赖性RNA聚合酶在微孔板形式中展示了这种PETE检测方法的实用性，以延伸自引发发夹RNA。PETE检测方法为筛选可能抑制聚合酶核酸结合或延伸活性的化合物提供了一种有效的方法。
