A high throughput molecular force assay for protein-DNA interactions
A high throughput molecular force assay for protein-DNA interactions
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DOI：
10.1039/c0lc00302f
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发表时间：
2011-01-01
期刊：
影响因子：
6.1
通讯作者：
Gaub, Hermann E.
中科院分区：
文献类型：
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作者：
Severin, Philip M. D.;Ho, Dominik;Gaub, Hermann E.
蛋白质-DNA相互作用（如转录因子结合）的准确和全基因组表征对现代生物学至关重要。强大的筛选方法出现了。但是这些技术中的绝大多数依赖于针对目标配体的特殊标记或标志物，而且它们中的大多数不适合检测低亲和力结合剂。在这篇文章中，分子力分析的基础上测量比较生物分子的解结合力的蛋白质-DNA相互作用的检测。结合力或非结合力的测量在生物学应用中具有几个独特的优点，因为检测的是某些分子之间的相互作用，而不仅仅是其中一个分子的存在。不需要针对蛋白质的标记或标志物，并且仅检测特异性结合的配体。此外，基于力的测定允许在拥挤和不透明的周围环境中检测在广泛的亲和力范围内的配体。我们证明，分子力测定允许高灵敏度和快速检测蛋白质-DNA相互作用。作为原理的证明，测量蛋白质EcoRI与其DNA识别序列的结合，并确定亚纳摩尔范围内的相应解离常数。此外，我们引入了一个新的，简化的设置采用FRET对的分子水平和标准的落射荧光读出。由于这些进步，我们现在可以证明，几微米的特征尺寸足以用于测量过程。这将在高通量筛选中开辟一个新的范例，具有基于力的配体检测的所有优点。
An accurate and genome-wide characterization of protein-DNA interactions such as transcription factor binding is of utmost importance for modern biology. Powerful screening methods emerged. But the vast majority of these techniques depend on special labels or markers against the ligand of interest and moreover most of them are not suitable for detecting low-affinity binders. In this article a molecular force assay is described based on measuring comparative unbinding forces of biomolecules for the detection of protein-DNA interactions. The measurement of binding or unbinding forces has several unique advantages in biological applications since the interaction between certain molecules and not the mere presence of one of them is detected. No label or marker against the protein is needed and only specifically bound ligands are detected. In addition the force-based assay permits the detection of ligands over a broad range of affinities in a crowded and opaque ambient environment. We demonstrate that the molecular force assay allows highly sensitive and fast detection of protein-DNA interactions. As a proof of principle, binding of the protein EcoRI to its DNA recognition sequence is measured and the corresponding dissociation constant in the sub-nanomolar range is determined. Furthermore, we introduce a new, simplified setup employing FRET pairs on the molecular level and standard epi-fluorescence for readout. Due to these advancements we can now demonstrate that a feature size of a few microns is sufficient for the measurement process. This will open a new paradigm in high-throughput screening with all the advantages of force-based ligand detection.