DNA Bubble Formation and Kinetics Studies by Single-Molecule FRET

Harvard University, Cambridge MA

Investigators

Abstract

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.