RUI: Elucidating the mechanisms of site specific DNA cleavage using single molecule methods

该项目将研究蛋白质分子如何能够准确快速地找到基因组中的特定位置。由于人类基因组中有超过30亿个碱基对，这就像大海捞针一样。科学家们在搜索过程中发现，许多蛋白质沿着DNA滑动。该项目将使用可以观察DNA单分子的高灵敏度技术，研究蛋白质滑动的方式和原因。在这个项目中研究的蛋白质被称为限制性内切酶，一旦它们与目标位点结合，就会将DNA切割成两条链。许多调节和维持基因组的蛋白质在其正常功能中切割DNA。新的基因工程技术也需要在特定的位点切割DNA。这个项目将调查这个过程是如何发生的。所有的实验室工作都将由本科生进行，他们将接受尖端技术技能、科学思维和解决问题能力的培训。该研究的概念将被纳入研究者讲授的课程。此外，调查员将为波士顿公立学校的学生和教师举办讲习班。这些研讨会将让高中生接触到科学研究，并帮助教师制作可以带回去在课堂上使用的材料。位点特异性DNA切割是抗体产生、原核适应性免疫系统和基因组编辑的关键步骤。本项目将使用单分子技术和计算建模相结合的方法研究模型系统（限制性内切酶）的目标搜索策略和切割机制。在一项技术中，与DNA连接的微珠将用于测量单个DNA分子切割的精确时间。在一次实验中，可以测量数百个DNA，以产生高分辨率的动力学数据。第二种技术将使用荧光来追踪单个蛋白质沿着DNA的扩散。一维扩散常数，以及偏离率，可以从单粒子轨迹确定。将开发目标搜索的理论模型，并与实验数据进行比较。随机漫步的计算机模拟，以及基于化学动力学的模型，将被探索。该项目将培养本科生的研究、科学传播和科学写作能力。生物化学课程将引入系留布朗运动和DNA切割的模块。在拓展活动中，将为波士顿地区的高中生举办半天的研究体验活动。此外，还将为高中教师开设专业发展课程。

This project will investigate how protein molecules are able to find specific locations in the genome accurately and quickly. Since there are over three billion base pairs in the human genome, this is akin to finding a needle in a haystack. Scientists have discovered that many proteins slide along the DNA during their search. Using highly sensitive techniques that can observe single molecules of DNA, this project will investigate how and why proteins slide. The proteins studied in this project, known as restriction endonucleases, cleave DNA into two strands once they bind to their target site. Many proteins that regulate and maintain the genome cleave DNA in their normal functioning. New techniques of genetic engineering also require cleavage of DNA at a specific site. This project will investigate how this process occurs. All laboratory work will be carried out by undergraduate students who will be trained in cutting edge technical skills as well as in scientific thinking and problem solving. Concepts from this research will be included in courses taught by the investigator. In addition, the investigator will hold workshops for students and teachers from Boston public schools. The workshops will expose high school students to scientific research and help teachers to create materials they can take back with them to use in their classrooms.Site specific DNA cleavage is a crucial step in antibody production, the prokaryotic adaptive immune system and genome editing. This project will investigate the target search strategies and cleavage mechanisms of a model system (restriction endonucleases) using a combination of single-molecule techniques and computational modeling. In one technique, microbeads tethered with DNA will be used to measure the exact time of cleavage of individual DNA molecules. In a single experiment, hundreds of DNA can be measured to yield high resolution kinetic data. A second technique will use fluorescence to track the diffusion of individual proteins along DNA. One dimensional diffusion constants, as well as off-rates, can be determined from single particle trajectories. Theoretical models of target search will be developed and compared to experimental data. Computer simulations of random walks, as well as models based on chemical kinetics, will be explored. The PI will train undergraduate students in research, scientific communication and scientific writing. Modules on tethered Brownian motion and DNA cleavage will be introduced into a biochemistry course. In outreach activities, half day research experiences for Boston area high school students will be held. In addition, a professional development course for high school teachers will be created.