Department of Molecular Biology and Interdisciplinary Nanoscience Center, Aarhus University, 8000 Aarhus C, Denmark.
ACS Nano. 2009 Dec 22;3(12):4043-54. doi: 10.1021/nn9012912.
We previously demonstrated 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 was achieved by topoisomerase I mediated closure of a nicked DNA circle followed by rolling circle amplification leading to an anchored product that was visualized at the single molecule level by hybridization to fluorescently labeled probes (Stougaard et al. ACS Nano 2009, 3, 223-33). An important inherent property of the presented setup is, at least in theory, the easy adaptability to multiplexed enzyme detection simply by using differently labeled probes for the detection of rolling circle products of different circularized substrates. In the present study we demonstrate the specific detection of three different enzyme activities, human topoisomerase I, and Flp and Cre recombinase in nuclear extracts from human cells one at a time or multiplexed using the rolling circle amplification based single-molecule detection system. Besides serving as a proof-of-principle for the feasibility of the presented assay for multiplexed enzyme detection in crude human cell extracts, the simultaneous detection of Flp and Cre activities in a single sample may find immediate practical use since these enzymes are often used in combination to control mammalian gene expression.
我们之前证明了，在纳米尺寸范围内，拓扑异构酶 I 介导的单个 DNA 切割-连接事件可以转化为微米大小的 DNA 分子，这可以通过标准荧光显微镜很容易地检测到。这种转化是通过拓扑异构酶 I 介导的切口 DNA 环的闭合，然后进行滚环扩增，导致锚定产物，通过与荧光标记探针杂交在单分子水平上可视化（Stougaard 等人，ACS Nano 2009，3，223-33）。所提出的设置的一个重要固有特性是，至少在理论上，通过使用不同标记的探针来检测不同环形化底物的滚环产物，很容易适应多重酶检测。在本研究中，我们证明了使用滚环扩增的单分子检测系统，一次或多重检测来自人细胞的核提取物中的三种不同酶活性，即人类拓扑异构酶 I、Flp 和 Cre 重组酶。除了作为该检测方法在粗人细胞提取物中进行多重酶检测的可行性的原理验证之外，在单个样品中同时检测 Flp 和 Cre 活性可能会立即找到实际用途，因为这些酶经常组合使用来控制哺乳动物基因表达。
