A nonradioactive plate-based assay for stimulators of nonspecific DNA nicking by HIV-1 integrase and other nucleases.
A nonradioactive plate-based assay for stimulators of nonspecific DNA nicking by HIV-1 integrase and other nucleases.
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DOI：
10.1016/j.ab.2009.09.012
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发表时间：
2010-01-15
影响因子：
2.9
通讯作者：
Katzman M
中科院分区：
文献类型：
--
作者：
Sudol M;Tran M;Nowak MG;Flanagan JM;Robertson GP;Katzman M
逆转录病毒整合酶具有受某些化合物刺激的非特异性核酸内切酶活性，表明可以操纵整合酶来破坏病毒 DNA。为了鉴定整合酶刺激剂（IS）化合物作为潜在的抗病毒药物，我们开发了一种适合高通量筛选的非放射性测定方法。该测定使用 49 聚体寡核苷酸，其 5' 标记有荧光团，3' 标记有猝灭剂，旨在形成发夹，模拟基于凝胶的切口测定中的放射性双链底物。经过单次热变性并随后冷却至无切口底物和带切口产物的熔解温度之间的温度（无需循环）后，在实时 PCR 机器上分析 384 孔板中的反应。在这些条件下，未切口的 DNA 会重新形成发夹并淬灭荧光，而完全切口的 DNA 会产生大信号。该测定与时间、刺激剂浓度和整合酶的量呈线性关系，并且用于许多化学文库的溶剂浓度为 20% 不会干扰测定。该检测具有出色的 Z' 因子，并且能够可靠地检测已知的 IS 化合物。该测定适用于其他非特异性核酸酶，将有助于鉴定其他 IS 化合物，以开发刺激整合酶破坏逆转录病毒 DNA 的新型抗病毒策略。
Retroviral integrase enzymes have a nonspecific endonuclease activity that is stimulated by certain compounds, suggesting that integrase could be manipulated to damage viral DNA. To identify integrase stimulator (IS) compounds as potential antiviral agents, we have developed a nonradioactive assay that is suitable for high-throughput screening. The assay uses a 49-mer oligonucleotide that is 5'-labeled with a fluorophore, 3'-tagged with a quencher, and designed to form a hairpin that mimics radioactive double-stranded substrates in gel-based nicking assays. Reactions in 384-well plates are analyzed on a real-time PCR machine after a single heat denaturation and subsequent cooling to a point between the melting temperatures of unnicked substrate and nicked products (no cycling is required). Under these conditions, unnicked DNA reforms the hairpin and quenches fluorescence, whereas completely nicked DNA yields a large signal. The assay was linear with time, stimulator concentration, and amount of integrase, and 20% concentrations of the solvent used for many chemical libraries did not interfere with the assay. The assay had an excellent Z'-factor, and it reliably detected known IS compounds. This assay, which is adaptable to other nonspecific nucleases, will be useful for identifying additional IS compounds to develop the novel antiviral strategy of stimulating integrase to destroy retroviral DNA.
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影响因子：
56.9
作者：
Grimsby, J;Sarabu, R;Grippo, JF
通讯作者：
Grippo, JF
DOI：
10.1073/pnas.240460997
发表时间：
2000-12-05
影响因子：
11.1
作者：
Biggins, JB;Prudent, JR;Thorson, JS
通讯作者：
Thorson, JS
影响因子：
--
作者：
John, S;Fletcher, TM;Jonsson, CB
通讯作者：
Jonsson, CB
影响因子：
5.4
作者：
Schumann, G;Hermankova, M;Boeke, JD
通讯作者：
Boeke, JD
影响因子：
5.4
作者：
KATZMAN, M;SUDOL, M
通讯作者：
SUDOL, M