Single-Molecule Detection of Human Topoisomerase I Cleavage−Ligation Activity

详细信息    查看全文

• 作者：Magnus Stougaard ; Jakob S. Lohmann ; Angelo Mancino ; Süleyman Celik ; Felicie F. Andersen ; Jørn Koch ; Birgitta R. Knudsen
• 刊名：ACS Nano
• 出版年：2009
• 出版时间：January 27, 2009
• 年：2009
• 卷：3
• 期：1
• 页码：223-233
• 全文大小：483K
• 年卷期：0
• ISSN：1936-086X

文摘

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 assay 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.