Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing 100084, China.
Protein Cell. 2010 Mar;1(3):284-90. doi: 10.1007/s13238-010-0031-0. Epub 2010 Feb 24.
Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP(i)), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid high-throughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PP(i)-generated and ATP-free enzyme reactions.
目前,用于检测 HIV-RT(逆转录酶)活性的体外检测法需要放射性标记或化学修饰的核苷酸来检测反应产物。然而,这些检测法本质上是终点测量法,且劳动强度大。在此,我们描述了一种新颖的非放射性检测法,该方法基于焦磷酸测序偶联酶系统的原理,通过间接测量在新生链合成过程中产生的焦磷酸(PP(i))的释放量,来监测 HIV-RT 的活性。结果表明,我们的检测法具有高灵敏度,可用于快速高通量抗 HIV 治疗药物筛选,因为它速度快且方便。此外,该检测法可以以简单且灵敏的方式测量引物酶的活性,这表明这种新方法可以广泛用于分析 PP(i)产生和无 ATP 酶反应的活性。