Fluorescent observation of individual DNA molecules on phospholipid bilayers and its application to analysis of DNA-protein interactions

H. Kurita, T. Takata, H. Yasuda, K. Takashima, A. Mizuno
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Abstract

The advantage of single-molecule experiments on analysis of DNA-protein interactions is that it can manipulate the physical form of individual DNA molecules. In these experiments, surface treatments for anchoring individual DNA molecules are important. In this study, we carried out fluorescent observations of individual DNA molecules on phospholipid bilayers. First, we constructed a PDMS microchannel and neutravidin was immobilized on the glass surface to tether individual DNA molecules. And then, DOPC liposomes were applied to the microchannel and the lipid bilayers were constructed. Biotinylated DNA molecules labeled with fluorescent dyes were then injected to the channel and they were tethered to the glass surface. As a result, we obtained clear images of the DNA molecules. Intriguingly, in addition, we found that if the fluorescent observation was carried out with the phospholipid bilayers, photobleaching of the fluorescently-labeled DNA was slower by comparison with that without the bilayers. Moreover, we applied this method to DNA-protein interactions at a single-molecule level.
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磷脂双层上单个DNA分子的荧光观察及其在DNA-蛋白质相互作用分析中的应用
单分子实验分析DNA-蛋白质相互作用的优势在于它可以操纵单个DNA分子的物理形态。在这些实验中,锚定单个DNA分子的表面处理是重要的。在这项研究中,我们对磷脂双层上的单个DNA分子进行了荧光观察。首先,我们构建了PDMS微通道,并将中性蛋白固定在玻璃表面以系住单个DNA分子。然后将DOPC脂质体应用于微通道,构建脂质双分子层。然后,用荧光染料标记的生物素化DNA分子被注射到通道中,并被拴在玻璃表面。结果,我们获得了DNA分子的清晰图像。有趣的是,另外,我们发现,如果用磷脂双分子层进行荧光观察,荧光标记的DNA的光漂白速度比没有双分子层的DNA慢。此外,我们将这种方法应用于单分子水平的dna -蛋白质相互作用。
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