Ligatable fluorescent probes using energy transfer for apoptosis detection

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DESCRIPTION (provided by applicant): This project will introduce a new approach for in situ detection of apoptotic cells. The approach will be applicable to fixed tissue samples and to live cell cultures. It will detect apoptosis driven by caspase- dependent and caspase-independent mechanisms. The approach will use fluorescence energy transfer (FET) oligoprobes to detect blunt ended DNA cuts with terminal 3'OH/5'PO4 produced by caspase-dependent and -independent apoptotic nucleases. These cuts represent a selective and general marker of apoptosis absent in necrosis. The FET probes will indicate successful detection of their target breaks by changing color. This new apoptosis labeling technology will make possible more sensitive apoptosis detection and discrimination between programmed and non-programmed cell death. The Specific Aims of the proposal are: 1) To develop a new and general apoptosis detection technology using fluorescence energy transfer probes and T4 DNA ligase, which will selectively label a specific type of blunt-ended DNA breaks common for various apoptotic executioner nucleases. To test the probes in fixed tissue sections using several models of apoptosis and necrosis. 2) To expand the methodology developed in the Aim 1 to live cell cultures. To verify selective labeling of different types of apoptotic cell death and its discrimination from necrosis using cell culture models of apoptosis and necrosis. 3) To validate the new approach by applying it to study focal cerebral ischemia as a condition where necrosis and several types of apoptosis are simultaneously present. To investigate the initiation and dynamics of apoptotic and necrotic cell death in brain after experimental stroke in rats. The proposed research will introduce the new enabling technology important for a wide range of clinical and research studies. Its application to brain ischemia will provide information essential for the development of precise and effective therapeutic interventions. PUBLIC HEALTH RELEVANCE: The proposed project will result in the development of a new assay for the needs of medical diagnostics and pathology. The technology will allow precise evaluation of the effects of therapy in diseases where cell death and DNA damage have prognostic value, such as stroke, Alzheimer's disease, and various cancers.