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，因此类似于在干草堆中找到针头。科学家发现，许多蛋白质在搜索过程中沿着DNA滑动。使用高度敏感的技术可以观察到DNA的单分子，该项目将研究蛋白质的滑动方式和原因。该项目中研究的蛋白质（称为限制性核酸内切酶）一旦结合到目标位点，将DNA切成两个链。许多蛋白质在正常功能中调节和维持基因组切割DNA的蛋白质。基因工程的新技术还需要在特定部位切割DNA。该项目将研究此过程的发生方式。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。这项研究的概念将包括在调查员教授的课程中。此外，调查人员还将为波士顿公立学校的学生和老师举办研讨会。这些研讨会将使高中学生接触科学研究，并帮助教师创建可以随身携带的材料，以便在课堂上使用。当地特定的DNA裂解是抗体生产，原核生物适应性免疫系统和基因组编辑的关键一步。该项目将使用单分子技术和计算建模的组合研究模型系统（限制性核酸内切酶）的目标搜索策略和裂解机制。在一种技术中，将使用DNA束缚的微粒来测量单个DNA分子切割的精确时间。在一个实验中，可以测量数百个DNA以产生高分辨率动力学数据。第二种技术将使用荧光来跟踪单个蛋白沿DNA的扩散。一维扩散常数以及离速率可以从单个粒子轨迹确定。将开发目标搜索的理论模型并将其与实验数据进行比较。将探索随机步行的计算机模拟以及基于化学动力学的模型。 PI将培训本科生的研究，科学沟通和科学写作。束缚的布朗运动和DNA裂解的模块将被引入生物化学过程中。在外展活动中，将举办波士顿地区高中生的半天研究经验。此外，还将为高中教师创建专业发展课程。