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

RUI：利用单分子方法阐明位点特异性 DNA 切割的机制

基本信息

批准号：
1715317
负责人：
Allen Price
金额：
$ 34.05万
依托单位：
Emmanuel College
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1715317&HistoricalAwards=false
关键词：
RUI Elucidating mechanisms site specific

项目摘要

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.

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，这类似于大海捞针。科学家们发现，在他们的搜索过程中，许多蛋白质会沿着DNA滑动。使用可以观察DNA单分子的高灵敏度技术，该项目将研究蛋白质如何以及为什么滑动。在这个项目中研究的蛋白质，被称为限制性内切核酸酶，一旦它们结合到它们的靶位点，就将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。基因工程的新技术也需要在特定位点切割DNA。本项目将研究这一过程是如何发生的。所有实验室工作将由本科生进行，他们将接受尖端技术技能以及科学思维和解决问题的培训。本研究的概念将纳入研究者教授的课程中。此外，调查员将为波士顿公立学校的学生和教师举办研讨会。这些工作坊将让高中生接触科学研究，并帮助教师制作可以带回课堂使用的材料。位点特异性DNA切割是抗体生产、原核适应性免疫系统和基因组编辑的关键步骤。本项目将结合单分子技术和计算建模，研究模型系统（限制性内切酶）的目标搜索策略和切割机制。在一种技术中，用DNA拴系的微珠将用于测量单个DNA分子切割的确切时间。在单个实验中，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跟踪单个蛋白质沿着DNA的扩散。一维扩散常数，以及解离速率，可以从单粒子轨迹确定。目标搜索的理论模型将开发和实验数据进行比较。将探讨随机行走的计算机模拟以及基于化学动力学的模型。 PI将在研究，科学交流和科学写作方面培养本科生。生物化学课程将引入有关束缚布朗运动和DNA切割的模块。在外展活动中，将为波士顿地区的高中生举办半天的研究体验。此外，还将为高中教师开设专业发展课程。