) A new approach for visualization and detailed analysis of DNA damage at the level of the single cell will be developed. The approach will be based on in situ ligation of double-stranded and hairpin shaped DNA probes with specific architectures that will bind to the ends of DNA in tissue sections. The probes will be designed to selectively detect various types of double strand breaks in cellular DNA. They will be used to find a specific marker of apoptotic DNA damage in order to distinguish it from DNA damage in necrosis, as well as from potentially reparable DNA damage in cells showing no morphological signs of apoptosis or necrosis. The probes will be tested in different models of apoptosis and nonapoptotic DNA damage. The methodology will also be used to study focal brain ischemia, to determine the percentage of different types of DNA damage in a variety of important brain regions. This determination will permit better assessment of cellular damage after ischemia onset, and will be helpful for development of better therapies for ischemic brain damage, as well as for the other CNS diseases characterized by various types of DNA damage. The proposed technology will allow simultaneous evaluation of several types of alterations in DNA at the level of single cell. It, therefore, can be used in molecular and cell biology research, as well as by clinicians in many fields, including pathology oncology, cardiology and the clinical neurosciences.
