High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging.
High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging.
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DOI：
10.1021/acs.langmuir.5b02416
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发表时间：
2015-09-22
期刊：
影响因子：
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通讯作者：
Finkelstein IJ
中科院分区：
文献类型：
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作者：
Gallardo IF;Pasupathy P;Brown M;Manhart CM;Neikirk DP;Alani E;Finkelstein IJ
蛋白质-DNA相互作用的单分子研究为DNA新陈代谢的几乎每一个方面的分子机制提供了关键的见解。DNA幕是一种在液体脂质双层上组织DNA分子阵列的方法，它的发展增加了在单个实验中可以观察到的反应数量，极大地促进了这些研究。然而，DNA窗帘的用途受到在石英显微镜载玻片上沉积纳米级脂类扩散屏障的挑战的限制。在这里，我们描述了一种基于UV光刻的方法，用于大规模制造铬(Cr)特征以及在这些特征处DNA分子的组织，以用于高通量单分子研究。我们通过在单个微流控流动池中组装792个独立的DNA阵列(包含90万个DNA分子)来演示这种方法。作为原理的第一个证据，我们跟踪了MLH1-MLH3的扩散，这是一种参与DNA错配修复和减数分裂重组的异二聚体复合体。为了进一步强调这种方法的实用性，我们展示了一种双通道流通池，它有助于在不同的DNA底物上同时进行实验。我们的技术极大地减少了与组装DNA窗帘相关的挑战，并为从单个单分子实验中快速获取大型统计数据集铺平了道路。
Single-molecule studies of protein–DNA interactions have shed critical insights into the molecular mechanisms of nearly every aspect of DNA metabolism. The development of DNA curtains—a method for organizing arrays of DNA molecules on a fluid lipid bilayer—has greatly facilitated these studies by increasing the number of reactions that can be observed in a single experiment. However, the utility of DNA curtains is limited by the challenges associated with depositing nanometer-scale lipid diffusion barriers onto quartz microscope slides. Here, we describe a UV lithography-based method for large-scale fabrication of chromium (Cr) features and organization of DNA molecules at these features for high-throughput single-molecule studies. We demonstrate this approach by assembling 792 independent DNA arrays (containing >900 000 DNA molecules) within a single microfluidic flowcell. As a first proof of principle, we track the diffusion of Mlh1-Mlh3—a heterodimeric complex that participates in DNA mismatch repair and meiotic recombination. To further highlight the utility of this approach, we demonstrate a two-lane flowcell that facilitates concurrent experiments on different DNA substrates. Our technique greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large statistical data sets from individual single-molecule experiments.