RUI: Developing Models of Facilitated Diffusion for DNA Binding Proteins

RUI：开发 DNA 结合蛋白易化扩散模型

• 批准号: 1205814
• 负责人: Allen Price
• 金额: $ 23.29万
• 依托单位: Emmanuel College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1205814&HistoricalAwards=false
• 关键词: RUI; Developing; Models; Facilitated; Diffusion

In this project, the PI will study how proteins that bind to specific sequences in DNA find their target sequences. It is known that some DNA binding proteins first bind non-specifically to DNA and then diffuse one-dimensionally along the DNA until they find their target sequence. The name "facilitated diffusion" is used to describe this process. Although this behavior is thought to be generic, it has only been observed in a handful of systems. In addition, single molecule observations of this process often occur in complicated environments involving shear flow and non-equilibrium fluctuations in the DNA, conditions which have not been adequately included in quantitative models. The PI will use two single molecule experimental techniques he has developed in order to detect and characterize facilitated diffusion of type II restriction endonucleases, enzymes which cleave double stranded DNA. The first technique is a flow stretching assay, in which a surface immobilized DNA is stretched out by fluid drag forces on a bead which is attached to the free end of the DNA. The second technique employs single molecule TIRF imaging of fluorescently labeled proteins diffusing one-dimensionally along tethered DNAs. Numerical simulations will be employed to explain the experimental results. The PI has developed a numerical simulation of tethered DNAs in shear flow. This simulation will be extended to include diffusing DNA binding proteins, and the numerical model developed will be validated against experimental results. This project will produce more realistic models of in vitro facilitated diffusion which will assist in translating experimental results in these systems to biologically relevant conditions. Additionally, the project will contribute to the understanding of reaction-diffusion behavior in flow conditions encountered in microfluidic cells, a problem relevant to lab-on-a-chip applications. Performing this research at an undergraduate institution, the PI will train and employ undergraduate students in his lab. These students will be mentored in research, in scientific presentation skills (through poster presentations at scientific meetings) and in scientific writing (through authorship on publications resulting from this project). In addition, the PI has developed a simplified DNA tethering protocol for use in pedagogy. Two experimental modules for use in laboratory courses will be developed that use this method. The first will be implemented in the PI's introductory physics course, and will examine tethered Brownian motion and the mechanical properties of DNA. The second will be used in an introductory molecular biology course, and will focus on the biophysics of restriction endonucleases. The PI will create a summer workshop for high school students. In this workshop, students will learn concepts from single molecule biological physics and participate in a hands-on demonstration of DNA tethering. The PI will also create a professional development course for high school teachers. In this course, teachers will learn how to perform DNA tethering experiments they can do in their classrooms. Teachers will receive materials and supplies for the experiments and the PI will visit their classrooms for demonstrations and to speak with students about single molecule biological physics.

在该项目中，PI将研究与DNA中特定序列结合的蛋白质如何找到其靶序列。众所周知，某些DNA结合蛋白首先非特异性与DNA结合，然后沿DNA散射一维，直到找到目标序列。 “促进扩散”的名称用于描述此过程。尽管这种行为被认为是通用的，但仅在少数系统中才观察到它。此外，该过程的单分子观察通常发生在涉及剪切流和非平衡波动的复杂环境中，在DNA中，这些条件尚未充分包含在定量模型中。 PI将使用他开发的两种单分子实验技术，以检测和表征II型限制性核酸内切酶的促进扩散，即裂解双链DNA的酶。第一种技术是一种流伸缩测定法，其中固定的DNA的表面通过珠子上的流体阻力拉伸，该珠子附着于DNA的自由端。第二种技术采用单分子TIRF成像，沿着系带DNA散射一维的荧光标记的蛋白质。数值模拟将用于解释实验结果。 PI在剪切流中开发了束缚DNA的数值模拟。该模拟将扩展到包括扩散DNA结合蛋白的扩散，并且开发的数值模型将得到实验结果的验证。该项目将产生更现实的体外促进扩散模型，这将有助于将这些系统中的实验结果转化为具有生物学相关条件。此外，该项目将有助于理解微流体细胞中遇到的流动条件下的反应扩散行为，这是与实验室中芯片应用相关的问题。 PI在本科机构进行这项研究，将培训并雇用本科生在他的实验室中。这些学生将在研究，科学演示技巧（通过科学会议上的海报演讲）和科学写作（通过该项目的出版物的作者身份）中获得指导。此外，PI开发了一种简化的DNA绑定方案，以用于教学法。将开发两个用于实验室课程的实验模块，用于使用此方法。第一个将在PI的介绍性物理课程中实施，并将检查束缚的布朗运动和DNA的机械性能。第二个将用于介绍性分子生物学课程，并将重点放在限制性核酸内切酶的生物物理学上。 PI将为高中生创建一个夏季研讨会。在这个研讨会中，学生将从单分子生物物理学中学习概念，并参加动手演示DNA绑扎。 PI还将为高中教师创建专业发展课程。在本课程中，老师将学习如何在课堂上进行他们可以进行的DNA绑扎实验。教师将收到实验的材料和用品，PI将访问其教室进行演示，并与学生谈论单分子生物物理学。