DNA Bubble Formation and Kinetics Studies by Single-Molecule FRET

这个项目的目的是实验表征热诱导（自发）DNA气泡形成的动力学。气泡动力学（打开和关闭时间）将在特定的DNA序列中直接测量，并研究温度、离子强度和蛋白质与单链部分结合对气泡稳定性的影响。实验将在单分子水平上使用荧光共振能量转移（FRET）来检测气泡形成和气泡关闭的时间尺度，这些时间尺度从亚毫秒到几秒不等，迄今为止还没有被探索过。这项工作是基于最近开发的一种高通量单分子成像仪器，以及一种用FRET对对DNA发夹分子进行内部标记的方案。该项目将涉及多个教育组成部分。首席研究员的实验室为研究生和本科生参与研究的所有阶段做好了准备，从样品制备、单分子数据采集到数据处理。学生将来自多个专业，生物物理学、分子生物学和应用物理学。与理论小组的合作将使更多的学生接触到研究工作。这个项目的资金由数学和物理科学理事会物理司和多学科活动办公室以及生物科学理事会分子和细胞生物学司提供。

The aim of this project is to experimentally characterize the kinetics of thermally induced (spontaneous) DNA bubble formation. The bubble kinetics (opening and closing times) will be directly measured in specific DNA sequences, and the effects on the bubble's stability of temperature, ionic strength, and protein binding to the single-stranded parts will be studied. The experiments will use Fluorescence Resonance Energy Transfer (FRET) at the single molecule level to detect bubble formation and bubble closure for a wide range of timescales that have not been explored so far, from sub-milliseconds to many seconds. The work is based on the recent development of an instrument for high-throughput single-molecule imaging, and a protocol for the internal labeling of DNA hairpin molecules with FRET pairs.The project will involve multiple educational components. The Principal Investigator's laboratory has prepared the ground for the participation of students at the graduate and undergraduate level in all stages of the research, from sample preparation, single molecule data acquisition, to data processing. Students will come from multiple programs, biophysics, molecular biology, and applied physics. The collaboration with theoretical groups will expose even more students to the research effort. Funding for this project is provided by the Division of Physics and the Office of Multidisciplinary Activities in the Mathematical and Physical Sciences Directorate and by the Division of Molecular and Cellular Biology in the Directorate for Biological Sciences.