A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity
A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity
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DOI：
10.1016/j.ab.2007.03.039
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发表时间：
2007-06-15
影响因子：
2.9
通讯作者：
Peersen, Olve B.
中科院分区：
文献类型：
--
作者：
Mestas, Santano P.;Sholders, Aaron J.;Peersen, Olve B.
我们设计了一种简单的高通量筛选兼容的基于荧光偏振的测定，可用于检测核酸聚合酶的延伸活性。该测定使用5 '端标记的模板链，并且依赖于来自荧光标记的偏振信号的增加，因为它通过酶的作用被拉向活性位点。如果寡核苷酸足够短，则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），并使用来自脊髓灰质炎病毒的RNA依赖性RNA聚合酶以微量滴定板形式证明了该PETE测定的实用性，以延伸自引发发夹RNA。PETE测定提供了用于筛选可抑制聚合酶的核酸结合或延伸活性的化合物的有效方法。(c)2007爱思唯尔公司All rights reserved.
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. (c) 2007 Elsevier Inc. All rights reserved.