Single-Molecule Detection of Human Topoisomerase I Cleavage-Ligation Activity
Single-Molecule Detection of Human Topoisomerase I Cleavage-Ligation Activity
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DOI：
10.1021/nn800509b
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发表时间：
2009-01-01
期刊：
影响因子：
17.1
通讯作者：
Knudsen, Birgitta R.
中科院分区：
文献类型：
--
作者：
Stougaard, Magnus;Lohmann, Jakob S.;Knudsen, Birgitta R.
在目前的研究中，我们证明了单个人类拓扑异构酶I介导的DNA切割-连接事件在纳米尺度内发生到微米大小的DNA分子的转化，很容易用标准荧光显微镜检测到。这种转化是通过拓扑异构酶I介导的缺口DNA哑铃结构的闭合来实现的，然后是滚动圈扩增。所得产物由DNA哑铃的多个串联重复组成，随后可以通过退火到荧光标记探针来可视化。由于扩增不涉及热循环，每个荧光滚动圈产物，在显微镜分析时产生一个单独的信号，将对应于一个单一的人类拓扑异构酶I介导的裂解-连接事件。关于灵敏度、速度和性能的易用性，所呈现的活动为；基于单分子产物检测的方法优于目前使用超卷曲质粒或放射性标记寡核苷酸作为拓扑异构酶I活性底物的最先进的检测方法。此外，在实验设计中固有的是易于适应多路复用和/或高通量系统。人拓扑异构酶I是临床重要抗癌药物的细胞靶点，这些药物的作用直接对应于细胞内拓扑异构酶I的切割-连接活性水平。因此，我们认为，在给定样本中直接测量切割-结扎事件的数量，具有很大的诊断潜力，大大增加了拓扑异构酶I定向化疗治疗前准确预后的可能性。
In the present study, we demonstrate the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion is achieved by topoisomerase I mediated closure of a nicked DNA dumbbell structure, followed by rolling circle amplification. The resulting product consists of multiple tandem repeats of the DNA dumbbell and can subsequently be visualized by annealing to fluorescently labeled probes. Since amplification involves no thermal cycling, each fluorescent rolling circle product, which gives rise to an individual signal upon microscopic analysis, will correspond to a single human topoisomerase I mediated cleavage-ligation event. Regarding sensitivity, speed, and ease of performance, the presented activity as;ay based on single-molecule product detection is superior to current state of the art assays using supercoiled plasmids or radiolabeled oligonucleotides as the substrate for topoisomerase I activity. Moreover, inherent in the experimental design is the easy adaptation to multiplexed and/or high-throughput systems. Human topoisomerase I is the cellular target of clinically important anticancer drugs, and the effect of such drugs corresponds directly to the intracellular topoisomerase I cleavage-ligation activity level. We therefore believe that the presented setup, measuring directly the number of cleavage-ligation events in a given sample, has great diagnostic potential, adding considerably to the possibilities of accurate prognosis before treatment with topoisomerase I directed chemotherapeutics.