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

这个由生物传感/CBET项目授予的NSF奖项支持了波士顿大学于纽尔教授在高通量平台上研究DNA-蛋白质复合体的构象和动力学的工作。DNA功能和DNA修复依赖于与蛋白质-组蛋白、转录蛋白质和它们之间的核酸酶的相互作用。基因转录的调节涉及形成特定的蛋白质-DNA复合体，这些复合体将DNA的遥远区域聚集在一起，并可以使DNA急剧弯曲或扭结。这些构象变化被认为在蛋白质识别DNA上的特定结合部位方面起着重要作用，因此，详细了解特定络合物的构象和形成动力学具有相当大的生物学意义。然而，目前还不存在直接测量这些生物分子构象所需的长度标尺。目前的荧光方法不能提供必要的分辨率，虽然电子显微镜具有高分辨率，但它会破坏样品。这项研究计划将开发和展示一种平台技术，用于精确定量测量DNA-蛋白质复合体在其自然环境中的构象。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的DNA阵列相结合。一种以高通量阵列形式精确测量DNA-蛋白质复合体形状的方法可以解决关于DNA-蛋白质复合体构象和取向的生物物理学的一系列重要问题。这一拟议计划的成功将为DNA-蛋白质复合体的结构测量提供一个独特的工具，显著降低成本和提高速度，并使研究基因调控和表达的基本原理成为可能。该项目的重大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在专业背景下通过日记俱乐部、暑期学校、研究研讨会以及参加当地、国内和国际会议来练习和完善沟通技能。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演讲，或者在暑期研究和沉浸式科学课程中与老师合作，或者在实验室研究中与城市高中生合作，以建立追求STEM职业生涯的动力。

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.