Using DNA Curtains to Reveal the Mechanisms of Target Site Location by DNA Binding Proteins

智力优势：该项目解决了生物学中的一个基本问题：位点特异性和结构特异性DNA结合蛋白如何在大量非特异性DNA中定位它们的靶标？这个问题将使用多学科的方法来研究，包括纳米工程、表面化学、物理和生物化学。蛋白质定位罕见靶点的能力对于基因表达、DNA复制、染色体动力学和基因组维持的各个方面都是必不可少的，但这些过程如何发生的有意义的细节通常是不可用的。本项目将重点关注复制后错配修复（MMR）作为模型系统，并将进行实验以精确确定MMR蛋白MutSa和MutLa如何定位和响应其特定的DNA靶标。尽管经过多年的深入研究，这些过程的详细机制尚不清楚，这主要是由于集合级生化测量的固有局限性。为了克服这些限制，该项目将利用纳米制造的“DNA窗帘”，并应用全内反射荧光显微镜（TIRFM）来直接可视化单个蛋白质复合物，因为它们在单个DNA分子上寻找并参与其目标位点。这种独特的单分子成像方法是由PI实验室首创的，通过对多个反应轨迹进行并行成像，可以从单个分子中实时快速收集统计相关信息。更广泛的影响-格林博士实验室开发的技术为实时、单分子核蛋白复合物分析提供了一种“高通量”方法。PI积极地将这项技术传播到更广泛的研究社区，因为它有可能为涉及蛋白质和DNA分子之间相互作用的几乎任何生物系统提供新的见解。这种跨学科的工作也为受训者提供了一个前沿的，广泛的教育经验，使他们能够在完成学位要求后追求重要的科学事业。为了促进对生物科学的单分子方法的理解，这些技术已被整合到大学的本科和研究生课程中。PI致力于共同努力，特别是通过研究型教育项目，促进年轻学生的科学训练和发展。他是哥伦比亚大学暑期本科生研究奖学金项目（SURF）的本科生导师，也是哈莱姆儿童协会的学生导师。该项目将把年轻的学生融入实验室科学工作的各个方面，从而为他们提供宝贵的、真实的研究经验。

Intellectual Merit: The project addresses a fundamental problem in biology: How do site- and structure-specific DNA-binding proteins locate their targets among a vast excess of nonspecific DNA? This question will be studied using a multi-disciplinary approach encompassing nanoscale engineering, surface chemistry, physics and biochemistry. The ability of proteins to locate rare targets is essential for all aspects of gene expression, DNA replication, chromosome dynamics and genome maintenance, yet meaningful details of how these processes occur are typically unavailable. This project will focus on post-replicative mismatch repair (MMR) as a model system, and experiments will be conducted to determine precisely how the MMR proteins MutSa and MutLa locate and respond to their specific DNA targets. Detailed mechanisms of these processes are not yet available, despite years of intensive investigation, largely due to the inherent limitations of ensemble-level biochemical measurements. To overcome these limitations, this project will utilize nanofabricated' DNA Curtains' and apply total internal reflection fluorescence microscopy (TIRFM) to directly visualize individual protein complexes as they search for and engage their target sites on individual molecules of DNA. This unique approach to single-molecule imaging was pioneered by the PI's laboratory, and enables rapid collection of statistically relevant information in real time from individual molecules by enabling parallel imaging of multiple reaction trajectories. Broader Impacts - The technology developed in Dr. Greene's laboratory provides a 'high-throughput' approach for real-time, single-molecule analysis of nucleoprotein complexes. The PI has actively disseminated this technology to the broader research community, as it has the potential to provide novel insights into virtually any biological system that involves interactions between protein and DNA molecules. This interdisciplinary work also provides trainees with a cutting-edge, broad-based educational experience that will allow them to pursue significant scientific careers upon completion of their degree requirements. To promote the understanding of single-molecule approaches to biological science, these technologies have been integrated into the University's undergraduate and graduate course curriculum. The PI is engaged in concerted efforts to advance the scientific training and development of younger students, particularly through research-oriented educational programs. He serves as an undergraduate student mentor for Columbia University's Summer Undergraduate Research Fellowship program (SURF), and as a student mentor for the Harlem Children's Society. The project will integrate younger students into all aspects of scientific work performed in the laboratory, thereby providing them with valuable, real-world research experiences.