Equipment for Spatiotemporal Dynamics of the Genome by 3D Orbital Tracking

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Equipment for Spatiotemporal Dynamics of the Genome by 3D Orbital Tracking Project Summary Our goal is to develop 3D orbital tracking, a powerful single-molecule fluorescence fluctuation technique, to measure the timing of gene activation, splicing, and genome organization at an active gene in living cells. By scanning a laser in a circle around a fluorescently labeled site of transcription in a confocal microscope, we can measure fluorescence intensity at high speed for a very long time in living cells. By fluorescent labeling, DNA, RNA, and protein factors in different colors, we can monitor complex assembly, transcription, splicing, and termination of RNA and the proximity of distal DNA elements like enhancers in the living genome. Looking at nature in a new way, or with a novel tool, often reveals previously unknown details. This proposal describes such a project. Although much work has been done to study transcription, splicing, and termination of RNA, they have yet to be able to characterize a complete kinetic profile of how the RNA is synthesized from a gene and then released or when splicing occurs. With such a tool, we may see much that was previously invisible. We can begin to discern complex biochemical reactions that can only be measured in living cells. This research project will support interdisciplinary undergraduate, graduate, and postdoctoral trainees, who will learn advanced techniques in genome biology and single-molecule imaging, shedding new light on the spatiotemporal dynamics of transcription and splicing. Project members will participate in activities to broaden the participation of underrepresented groups in science, mathematics, engineering, and technology.