An Electromagnetic Trapping Platform for Single Molecule Studies.

[摘要] Mechanical forces and torques play an important role in many biological processes ranging from gene transcription to protein conformational dynamics, and intracellular transportation.A range of spectroscopy techniques have been developed for direct measurements of force and torque applied to biomolecules, and fluorescence microscopy have been used extensively to study the dynamics of biomolecules and their interactions.However, to apply both force and torque simultaneously in electromagnetic tweezers, with two components decoupled and controlled independently, has not been accomplished.Also, the combination of torque spectroscopy with fluorescence microscopy has not been explored.The goal of this dissertation was to develop a novel magnetic trapping platform that is capable of simultaneously exerting and monitoring controlled forces and torques on single biomolecules via micrometer-sized magnetic particles while recording fluorescence images with high spatial and temporal resolution.Towards this goal, a system using electromagnets composed of a monopole that generates force, and a set of quadrupoles that generate torque, was integrated into an optical microscope.With this system, it is possible to apply tension in the piconewton force range to single molecules, and to independently modulate the trap torsional stiffness to twist molecules.Secondly, a novel method has been developed to directly track the angular displacement of optically anisotropic particles undergoing rotational motion using optoelectronic detectors.This method makes image acquisition and processing procedures commonly used in previous studies for detecting rotational motion unnecessary, and, when combined with the electromagnetic tweezers, can extend the bandwidth of control and detection of mechanical states of single molecules to the kilohertz regime.Finally, a dual-color, bifocal imaging system was implemented to allow detection of spectrally and spatially distinct fluorescent particles using a single camera with sub-pixel registration between two separate channels, and low mechanical drift.In conclusion, the advancement in the force/torque spectroscopy and fluorescence imaging techniques presented here holds significant potential in biological investigations that require fast control and detection of mechanical states of molecules and their dynamic properties.This system can be applied to studies of DNA-protein interactions, DNA conformation dynamics, and their dependence on the mechanical states of the DNA molecules.

[发布机构] University of Michigan

[效力级别] single molecule force spectroscopy

[关键词] electromagnetic tweezers;single molecule force spectroscopy;Mechanical Engineering;Science (General);Engineering;Science;Mechanical Engineering