Quantifying Single Molecule DNA-ligand Interactions

这个项目的目标是开发单分子方法来定量研究DNA与小分子和蛋白质的相互作用，并使用这些方法来研究特定的生物重要系统。研究人员将使用几种DNA结构(长短双链和单链DNA分子)和数据分析方法来探索DNA与配体相互作用的热力学和动力学，并确定随着蛋白质和小分子结合DNA结构随时间的变化。除了方法开发外，这项工作还将阐明DNA-小分子相互作用的基本生物物理，以及反转录转座子复制和DNA损伤反应中涉及的多种蛋白质相互作用的生物物理和生化机制。本项目涉及三个具体目标：1.利用单分子力谱研究DNA-小分子相互作用的结构动力学。2.探讨LINE1逆转录转座过程中蛋白质-DNA的动态相互作用。3.探讨在大肠杆菌中参与DNA聚合酶开关管理的蛋白质-蛋白质和蛋白质-DNA结合作用。PI将开发理解多种蛋白质与DNA相互作用的方法，并使用这些方法来探测DNA聚合酶管理中涉及的相互作用，这对细胞对DNA损伤的反应至关重要。PI开发了使用单分子操作来检查DNA结合的创新生物物理方法。PI将他的研究融入到教学实验方法中，他在培养研究生和本科生在物理、化学和生物相结合的跨学科研究方面有着良好的记录。除了定期聘用为期6个月的全日制物理合作社本科生外，他还招待了其他几所本科院校的本科生。PI正在接待一名教授和他的三名学生，他们来自一所以第一代大学生为主的地区性大学。这些经验将被用来扩大生物物理学的研究，并将生物物理学引入大学的课程。这位合作者是一所女子文理学院的教授，有着培养本科生女科学家的历史传统。她将让多名学生参与合成DNA结构和小分子复合体，他们将把它们带到东北大学，并用单分子力谱进行表征。该项目由分子和细胞生物科学部的分子生物物理组和物理部的生命系统物理学项目共同支持。

The goal of this project is to develop single molecule methods for the quantitative study of DNA interactions with small molecules and proteins and to use these methods to investigate specific biologically important systems. The investigators will use several DNA constructs (long and short double-stranded and single-stranded DNA molecules) and data analysis methods to probe the thermodynamics and kinetics of DNA-ligand interactions and determine the time-dependent changes of DNA structure as proteins and small molecules bind. In addition to method development, this work will shed light on the fundamental biophysics of DNA-small molecule interactions as well as the biophysical and biochemical mechanisms of multiple protein interactions involved in retrotransposon replication and DNA damage response. This project involves three specific aims: 1. To probe the structural dynamics of DNA-small molecule interactions using single molecule force spectroscopy. 2. To probe dynamic protein-DNA interactions important for LINE1 retrotransposition. 3. To probe the protein-protein and protein-DNA binding interactions that participate in the management of DNA polymerase switching in E. coli. The PIs will develop methods for understanding multiple protein interactions with DNA and use these methods to probe interactions involved in DNA polymerase management, which is critical for cellular responses to DNA damage. The PI has developed innovative biophysical methods for examining DNA binding using single molecule manipulation. The PI integrates his research into teaching experimental methods, and he has a strong track record of training graduate and undergraduate students in interdisciplinary research at the interface of physics, chemistry, and biology. In addition to regularly hiring full-time 6-month physics co-op undergraduate students, he has hosted undergraduates from several other undergraduate institutions. The PI is hosting a professor and three of his students from a regional university with a majority of first-generation college students. This experience will be used to expand biophysics research and introduce biophysics into the university's curriculum. The co-PI is a Professor at a liberal arts college for women with a historical tradition of training undergraduate women scientists. She will involve multiple students in synthesizing DNA constructs and small molecule complexes that they will bring to Northeastern and characterize by single molecule force spectroscopy. This project is jointly supported by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Physics Division.