High throughput quantification of conformation and kinetics of DNA-protein complexes

这项由生物传感/CBET项目颁发的NSF奖支持波士顿大学Ünlü教授在高通量平台上研究dna -蛋白质复合物的构象和动力学。DNA的功能和修复依赖于与蛋白质的相互作用，其中包括组蛋白、转录蛋白和核酸酶。基因转录的调控涉及特定蛋白质- DNA复合物的形成，这种复合物将DNA的遥远区域聚集在一起，并能使DNA急剧弯曲或扭结。这些构象变化被认为在蛋白质识别DNA上的特定结合位点中起重要作用，因此详细了解特定复合物的构象和形成动力学具有相当大的生物学意义。然而，目前的技术还无法获得直接测量这些生物分子构象所需的长度尺度。目前的荧光方法不能提供必要的分辨率，而电子显微镜具有高分辨率，它破坏了样品。该研究项目将开发并演示一种平台技术，用于精确定量测量dna -蛋白质复合物在其天然环境中的构象。我们的方法结合了波士顿大学（BU）和慕尼黑工业大学（TUM）的创新，将光学干涉技术与表面电场驱动的高度有序DNA阵列相结合。一种以高通量阵列格式精确测量dna -蛋白质复合物形状的方法可以解决一系列关于dna -蛋白质复合物构象和取向的生物物理学的重要问题。该计划的成功将提供一种独特的工具，显著降低成本，提高dna -蛋白质复合物结构测量的速度，并允许研究基因调控和表达的基本原理。这个项目的重要教育成果将是培养至少两名研究生。参与研究的学生将有机会通过期刊俱乐部、暑期学校、研究研讨会以及参加地方、国家和国际会议，在专业背景下练习和完善沟通技巧。他们将与来自德国和新加坡的国际合作者密切合作。学生还将获得向外行观众展示的经验和培训，最好的例子是在初中和高中课堂上展示，或与教师一起参加暑期研究和沉浸式科学课程，或与城市高中学生一起进行实验室研究，以建立追求STEM职业的动力。

0933670Unlu This NSF award by the Biosensing /CBET program supports work by Professor Ünlü at Boston University to study conformation and kinetics of DNA-protein complexes on a high-throughput platform. DNA function and DNA repair depend on interactions with proteins - histones, transcriptional proteins and nucleases among them. The regulation of gene transcription involves the formation of specific protein - DNA complexes that bring distant regions of DNA together and can sharply bend or kink the DNA. These conformational changes are postulated to play an important role in the recognition of specific binding sites on DNA by proteins and thus detailed understanding of the conformation and dynamics of formation of specific complexes are of considerable biological significance. Yet current technology does not exist to access the length scales necessary to directly measure these biomolecular conformation. Current fluorescence methods do not provide the necessary resolution and while the electron microscopy has high resolution it destroys the sample. This research program will develop and demonstrate a platform technology for the precise quantified measurement of conformation of DNA-protein complexes in their native environment. Our approach combines innovations from Boston University (BU) and Technical University of Munich (TUM), merging optical interference techniques with surface electric field driven, highly-ordered DNA arrays. A method that precisely measures the shape of DNA-protein complexes in a high-throughput array format can address a range of important questions about the biophysics of the conformation and orientation of DNA-protein complexes. The success of this proposed program will provide a unique tool with significant cost reduction and increase in speed in structural measurements of DNA-protein complexes and allow for the study the basic principles of regulation and expression of genes. The significant educational outcome of this program will be training at least two graduate students. The students involved in the research will have the opportunity to practice and perfect communication skills within a professional context through journal clubs, summer schools, research seminars, and participation in local, national, and international conferences. They will work closely with international collaborators from Germany and Singapore. Students also will have the experience and training of expositing to a lay audience, best exemplified by presenting in middle and high school classrooms, or working with teachers in summer research and immersive science classes, or with urban high school students in lab research to build motivation to pursue a STEM career.