RUI: Characterizing DNA target search using single molecule methods

RUI：使用单分子方法表征 DNA 靶标搜索

• 批准号: 2120878
• 负责人: Allen Price
• 金额: $ 37.88万
• 依托单位: Emmanuel College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2120878&HistoricalAwards=false
• 关键词: RUI; Characterizing; DNA; target; search

This project will investigate how proteins find specific locations in DNA. Since there are over three billion base pairs in the human genome, this is akin to finding a needle in a haystack. A variety of methods will be employed to investigate how proteins combine sliding, hoping and jumping along DNA into a search strategy. In one method, the time taken by the protein to find its target site will be measured. In another, the motion of individual proteins during search will be tracked in real time. Stretching or coiling the DNA during the search process will demonstrate how the different conformations of DNA in the cell affect the search. By varying the composition of the solution containing DNA and proteins, the effects of different cellular components can be determined. In another investigation, how so called "off-target" sites (sites that are similar to the target site and typically only vary by one or two base pairs) slow or perhaps even speed up the search will be studied. The laboratory work will be carried out by undergraduate students and concepts from this research will be included in courses taught by the principal investigator. In addition, the research team will hold summer day camps for students from elementary and middle schools in Boston, where these students will be exposed to research and the scientific method.This project will investigate search strategies of DNA binding proteins using single molecule methods. The model system chosen, restriction endonucleases, form part of the bacterial innate immune response and cleave foreign DNA at specific sites 4-8 base pairs in length. In one technique, micro beads tethered with DNA will be used to measure the exact time of cleavage of individual DNA molecules. Using this method, hundreds of DNAs can be measured in a single experiment to yield high resolution kinetic data. A second technique will use fluorescence to track the motion of individual proteins during search. One area of investigation will characterize the roles of sliding, hopping and jumping. The use of DNA roadblocks will distinguish between sliding, which will be blocked, and hopping, which can bypass roadblocks. Varying the conformation of DNA during single molecule imaging can test for the presence of enhanced jumping during coiling. Another area concerns off-target binding sites, which can slow target search, but also serve as reservoirs that can play a role in genetic regulation by transcription factors. By inclusion of star sites (non-cognate sites differing by a single base pair) in DNA, quantitative models of how the position and number of such sites affect search rates can be tested. A final area involves the role of macromolecular crowding. By introducing crowding agents, the effects both on target search rates (in bead tethering experiments) and on linear diffusion along DNA (in single molecule fluorescence tracking) can be addressed. Broader impacts of this project include training of undergraduate students, developing modules on tethered DNA for undergraduate courses, as well as a summer day camp for elementary and middle school students.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该项目将研究蛋白质如何在DNA中找到特定位置。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。将采用多种方法来研究蛋白质如何结合联合收割机滑动，希望和跳跃沿着DNA到一个搜索策略。在一种方法中，将测量蛋白质找到其靶位点所花费的时间。在另一种情况下，搜索过程中单个蛋白质的运动将被真实的跟踪。在搜索过程中拉伸或卷曲DNA将展示细胞中DNA的不同构象如何影响搜索。通过改变含有DNA和蛋白质的溶液的组成，可以确定不同细胞组分的影响。在另一项研究中，将研究所谓的“脱靶”位点（与靶位点相似且通常仅相差一个或两个碱基对的位点）如何减慢甚至加速搜索。实验室工作将由本科生进行，本研究的概念将纳入主要研究者教授的课程。此外，研究小组还将为波士顿的中小学生举办夏令营，让他们接触研究和科学方法。本项目将研究使用单分子方法搜索DNA结合蛋白的策略。选择的模型系统，限制性内切核酸酶，形成细菌先天免疫应答的一部分，并在长度为4-8个碱基对的特异性位点切割外源DNA。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。使用这种方法，可以在一个实验中测量数百个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搜索过程中单个蛋白质的运动。一个调查领域将描述滑动，跳跃和跳跃的作用。使用DNA路障将区分滑动，这将被阻止，跳跃，这可以绕过路障。在单分子成像期间改变DNA的构象可以测试在卷曲期间增强的跳跃的存在。另一个领域涉及脱靶结合位点，脱靶结合位点可以减缓目标搜索，但也可以作为水库，在转录因子的遗传调控中发挥作用。通过在DNA中包含星星位点（相差单个碱基对的非同源位点），可以测试此类位点的位置和数量如何影响搜索率的定量模型。最后一个领域涉及大分子拥挤的作用。通过引入拥挤剂，可以解决对目标搜索速率（在珠系实验中）和对沿着DNA的线性扩散（在单分子荧光跟踪中）的影响。该项目的更广泛的影响包括培养本科生，开发本科课程的拴系DNA模块，以及为中小学生举办夏令营。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。