RUI: Developing Models of Facilitated Diffusion for DNA Binding Proteins

在这个项目中，PI将研究与DNA中特定序列结合的蛋白质如何找到它们的目标序列。众所周知，一些DNA结合蛋白首先与DNA非特异性结合，然后沿着DNA一维扩散，直到找到它们的目标序列。这个过程被称为“促进扩散”。虽然这种行为被认为是通用的，但它只在少数系统中被观察到。此外，这一过程的单分子观察经常发生在涉及DNA剪切流动和非平衡波动的复杂环境中，这些条件尚未充分包括在定量模型中。PI将使用他开发的两种单分子实验技术来检测和表征II型限制性内切酶的促进扩散，这种酶可以切割双链DNA。第一种技术是流动拉伸试验，其中表面固定的DNA通过附着在DNA自由端上的头部上的流体阻力被拉伸。第二种技术采用单分子TIRF成像，荧光标记的蛋白质沿着拴着的dna一维扩散。数值模拟将被用来解释实验结果。PI开发了剪切流动中栓系dna的数值模拟。该模拟将扩展到包括DNA结合蛋白的扩散，并且开发的数值模型将根据实验结果进行验证。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生物学相关条件。此外，该项目将有助于理解微流体细胞在流动条件下的反应扩散行为，这是一个与芯片实验室应用相关的问题。在一个本科院校进行这项研究，PI将在他的实验室培训和雇用本科生。这些学生将在研究、科学展示技巧（通过在科学会议上的海报展示）和科学写作（通过在这个项目中发表论文）方面得到指导。此外，PI还开发了一种用于教学的简化DNA捆绑协议。将开发两个实验模块，用于使用这种方法的实验课程。第一个将在PI的入门物理课程中实施，并将检查系绳布朗运动和DNA的机械特性。第二个将用于分子生物学入门课程，并将重点放在限制性内切酶的生物物理学上。PI将为高中生创建一个暑期工作坊。在这个工作坊中，学生将学习单分子生物物理学的概念，并参与DNA系留的实际演示。PI还将为高中教师开设专业发展课程。在本课程中，教师将学习如何在课堂上进行DNA捆绑实验。教师将收到实验材料和用品，PI将访问他们的教室进行演示，并与学生讨论单分子生物物理学。

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.