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靶序列。已知一些DNA结合蛋白首先非特异性结合DNA，然后沿DNA一维沿着扩散，直到它们找到它们的靶序列。“易化扩散”这个名称用来描述这个过程。虽然这种行为被认为是普遍的，但它只在少数系统中被观察到。此外，这一过程的单分子观测往往发生在复杂的环境中，涉及剪切流动和DNA中的非平衡波动，这些条件尚未充分纳入定量模型。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（切割双链DNA的酶）的易化扩散。第一种技术是流动拉伸测定，其中表面固定的DNA通过附着于DNA自由端的珠上的流体拖曳力拉伸。第二种技术采用荧光标记蛋白质的单分子TIRF成像，所述荧光标记蛋白质沿沿着束缚的DNA一维扩散。数值模拟将被用来解释实验结果。PI开发了剪切流中系留DNA的数值模拟。该模拟将扩展到包括扩散DNA结合蛋白，并将对实验结果验证开发的数值模型。该项目将产生更现实的体外易化扩散模型，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中遇到的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，在科学演示技巧（通过在科学会议上的海报演示）和科学写作（通过对本项目产生的出版物的作者身份）的指导。此外，PI还开发了一个简化的DNA系留协议，用于教学。两个实验模块用于实验室课程将开发使用这种方法。第一个将在PI的入门物理课程中实施，并将研究拴系布朗运动和DNA的机械特性。第二个将用于分子生物学的入门课程，并将集中在限制性内切核酸酶的生物物理学。PI将为高中生创建一个夏季研讨会。在这个研讨会中，学生将学习单分子生物物理学的概念，并参加DNA拴系的实践演示。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拴系实验。教师将收到实验材料和用品，PI将参观他们的教室进行演示，并与学生谈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.