A continuous assay for DNA cleavage: The application of "break lights" to enediynes, iron-dependent agents, and nucleases
A continuous assay for DNA cleavage: The application of "break lights" to enediynes, iron-dependent agents, and nucleases
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DOI：
10.1073/pnas.240460997
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发表时间：
2000-12-05
影响因子：
11.1
通讯作者：
Thorson, JS
中科院分区：
文献类型：
--
作者：
Biggins, JB;Prudent, JR;Thorson, JS
尽管人们已经做了大量的努力来了解烯二炔切割DNA的机制，但仍然缺乏对这种现象的连续测定。事实上，除了DNA酶的测定外，大多数DNA切割事件的连续测定是不可用的。本文介绍了应用“分子断裂光”（一种单链寡核苷酸，采用茎环结构，在茎中带有5'荧光部分、3'非荧光猝灭部分和适当的切割位点）建立了第一个连续测定烯二炔切割DNA的方法。此外，通过使用所描述的测定直接比较天然存在的烯二炔类（加利车霉素和埃斯波霉素）、非烯二炔类小分子试剂（博来霉素、甲基丙基-EDTA-Fe（lt））的DNA切割，证明了该方法的通用性。和EDTA-Fe（II））。以及限制性内切酶BamHI。考虑到这种方法的简单性、速度和灵敏度，所描述的方法可以容易地扩展到高通量形式，并成为现代药物发现中筛选新的基于蛋白质或小分子衍生的DNA切割剂的新方法。
Although extensive effort has been applied toward understanding the mechanism by which enediynes cleave DNA, a continuous assay for this phenomenon is still lacking. In fact, with the exception of assays for DNase, continuous assays for most DNA cleavage events are unavailable. This article describes the application of "molecular break lights" (a single-stranded oligonucleotide that adopts a stem-and-loop structure and carries a 5' fluorescent moiety, a 3'-nonfluorescent quenching moiety, and an appropriate cleavage site within the stem) to develop the first continuous assay for cleavage of DNA by enediynes. Furthermore, the generality of this approach is demonstrated by using the described assay to directly compare the DNA cleavage by naturally occurring enediynes [calicheamicin and esperamicin), non-enediyne small molecule agents (bleomycin, methidiumpropyl-EDTA-Fe(lt). and EDTA-Fe(II]). as well as the restriction endonuclease BamHI. Given the simplicity, speed, and sensitivity of this approach, the described methodology could easily be extended to a high throughput format and become a new method of choice in modern drug discovery to screen for novel protein-based or small molecule-derived DNA cleavage agents.