Zhang Xirui, Daaboul George G, Spuhler Philipp S, Dröge Peter, Ünlü M Selim
Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.
Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA and Department of Electrical and Computer Engineering, Boston University, Boston, Massachusetts 02215, USA.
Nanoscale. 2016 Mar 14;8(10):5587-98. doi: 10.1039/c5nr06785e.
DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.
DNA结合蛋白在基因组的维持和功能中发挥着关键作用，然而，它们的特异性结合机制尚未完全被理解。最近，人们发现DNA结合蛋白通过识别和捕获DNA构象灵活性及变形，利用间接读出机制识别特定结合位点以执行其功能。基于高通量DNA微阵列的方法可提供大规模蛋白质-DNA结合信息，已显示出对蛋白质-DNA结合亲和力进行有效且全面的分析，但无法提供特定蛋白质-DNA复合物中DNA构象变化的信息。基于DNA微阵列的高通量能力，我们展示了一种定量方法，该方法能以微阵列形式同时测量与DNA结合的蛋白量以及由蛋白结合诱导的纳米级DNA构象变化。这两种测量均依赖于使用单一光学仪器以两种不同模式对分层底物进行光谱干涉测量。在第一种模式中，我们使用无标记光谱反射率技术对每个DNA斑点中与表面固定DNA结合的蛋白量进行定量，该技术可精确测量积累在底物上的蛋白和DNA的表面密度。在第二种模式中，对于每个DNA斑点，我们使用荧光垂直切片技术同时测量DNA构象变化，该技术可确定标记到表面固定DNA特定核苷酸上的荧光团的平均轴向高度。本文提出的方法与当前基于高通量DNA微阵列的技术相结合，有潜力成为一种快速且简单的方法，用于对构象特异性蛋白质-DNA相互作用进行定量和大规模表征。
