Single-Molecule Imaging of Biorecognition Kinetics

国家科学基金会化学部的化学测量和成像（CMI）计划支持这个项目。Joel M.哈里斯和他在犹他州大学的研究小组正在开发显微镜实验，以研究生物分子，特别是DNA的反应。DNA互补链之间的化学反应是生物体中遗传信息复制和转录的基本步骤。该项目的目标是开发显微镜方法，使DNA链之间的反应能够在单个分子在玻璃表面反应时在“电影”中观察到。了解这些反应对于开发用于疾病诊断的基于DNA的基因检测至关重要。单分子成像也是一种灵敏的检测方法，通过对单个分子进行计数，可以分析生物样本中极低浓度的靶DNA。更广泛的影响是通过培养研究生，本科生和博士后助理在显微镜和生物分析化学。这些主题与推进美国科学技术有关。该项目还通过为初中和高中学生开发显微镜下的化学练习来支持K-12科学教育和推广。该项目采用的独特方法是将未标记的DNA固定在捕获表面上，该捕获表面经过设计，与溶液中的荧光标记靶DNA的非特异性相互作用极低。其结果是一个高灵敏度的测量平台，其中捕获表面可以长时间使用，甚至可以在多个实验中重复使用。通过超分辨率成像，可以识别表面上的单个固定化DNA探针链，从而创建单分子捕获位点的微阵列。 该研究的影响包括对影响基于DNA的生物传感器性能的结构因素的理解。用于单分子成像的显微镜仪器的发展支持了几项合作，包括DNA适体发现，测量信号蛋白的膜亲和力，以及评估新的候选药物。

The Chemical Measurement and Imaging (CMI) program of the Division of Chemistry at the National Science Foundation supports this project. Professor Joel M. Harris and his research group at the University of Utah are developing microscopy experiments to investigate reactions of biological molecules, especially DNA. The chemical reaction between complementary strands of DNA is the fundamental step in duplication and transcription of genetic information in living organisms. The goal of this project is to develop microscopy methods that allow the reactions between DNA strands to be observed in "movies" of individual molecules as they react at a glass surface. Understanding these reactions is critical to the development of DNA-based genetic testing used in disease diagnosis. Single-molecule imaging also serves as a sensitive detection method, where counting individual molecules enables the analysis of extremely low concentrations of target DNA in biological specimens. The broader impacts are demonstrated through the training of graduate students, undergraduates, and postdoctoral associates in microscopy and bioanalytical chemistry. These topics are relevant to advancing U.S. science and technology. The project also supports K-12 science education and outreach through development of Chemistry-under-the-Microscope exercises for middle- and high-school students.The unique approach taken in this project is to immobilize unlabeled DNA on a capture surface that is engineered to have extremely low non-specific interactions with fluorescently-labeled target DNA in solution. The result is a high sensitivity measurement platform, where capture surfaces can be used for long periods of time, and even reused in multiple experiments. With super-resolution imaging, individual immobilized DNA probe strands on the surface can be identified, thereby creating a microarray of single-molecule capture sites. Impacts of the research include generating understanding of structural factors that influence the performance of DNA-based biosensors. The development of microscopy instrumentation for single-molecule imaging supports several collaborations including DNA aptamer discovery, measuring membrane affinity of signaling proteins, and assessing of new drug candidates.