Express assay for specific fluorescence imaging of apoptosis via phosphatase-assisted topoligation.

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Express assay for specific fluorescence imaging of apoptosis via phosphatase-assisted topoligation. Abstract Starting with DNA sequence obtained from Mimivirus we synthesized an unusual viral DNA topoisomerase. We found that the recombinant protein possesses extremely fast ligation activity. The topoisomerase can rapidly ligate DNA ends, finishing the reaction within several seconds after addition to DNA. This surpasses the speed of all known DNA ligases by two orders of magnitude. It permits a novel ultra-fast and specific assay for visualization of apoptotic cells in tissue sections. In this project we will develop a new express assay for in situ research. The assay will selectively label apoptotic, but not necrotic cells, based on detection of characteristic double-stranded DNA breaks produced by apoptotic executioner nucleases. At present these biomarkers are considered highly specific for programmed cell death, but their detection takes 24 hrs. The proposed technology will perform such detection within minutes. Now there are no assays with similar capability. The project will introduce a new ultra-fast bioimaging approach employing the unique properties of Mimivirus topoisomerase. It will close the technological gap and will create an advantageous labeling technique with a wide application field. The new quick and specific assay will be useful in biomedicine, particularly in apoptosis research, in express assessment of pathology samples and in studies where large-volume quantitations of programmed cell death cells are essential, such as in cancers, ischemic disorders, and degenerative diseases. The Specific Aims of the proposal are: 1) To develop the first express assay for specific detection of apoptosis in histological sections via labeling of blunt-ended 5’PO4 DNA breaks produced by apoptotic executioner nucleases. The assay will employ the novel phosphatase-assisted topoligation labeling technology. To test the new labeling approach using in vitro models. 2) To verify the new assay in several apoptotic models. To optimize its speed, sensitivity, specificity and applicability in fixed cells and tissue sections.