Ligatable fluorescent probes using energy transfer for apoptosis detection Abstract 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.
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.
|Minchew, Candace L; Didenko, Vladimir V (2014) Assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes. J Vis Exp :|
|Didenko, Vladimir V (2014) Selective detection of phagocytic phase of apoptosis in fixed tissue sections. Methods Mol Biol 1094:159-66|
|Minchew, Candace L; Didenko, Vladimir V (2014) Nanoblinker: Brownian motion powered bio-nanomachine for FRET detection of phagocytic phase of apoptosis. PLoS One 9:e108734|
|Minchew, Candace L; Didenko, Vladimir V (2014) Selective transport of cationized fluorescent topoisomerase into nuclei of live cells for DNA damage studies. Methods Mol Biol 1094:167-75|
|Minchew, Candace L; Didenko, Vladimir V (2012) In vitro assembly of semi-artificial molecular machine and its use for detection of DNA damage. J Vis Exp :e3628|
|Didenko, Vladimir V (2011) In situ ligation simplified: using PCR fragments for detection of double-strand DNA breaks in tissue sections. Methods Mol Biol 682:65-75|
|Didenko, Vladimir V (2011) 5'OH DNA breaks in apoptosis and their labeling by topoisomerase-based approach. Methods Mol Biol 682:77-87|
|Minchew, Candace L; Didenko, Vladimir V (2011) Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA. Molecules 16:4599-614|
|Hornsby, Peter J; Didenko, Vladimir V (2011) In situ ligation: a decade and a half of experience. Methods Mol Biol 682:49-63|
|Didenko, Vladimir V (2011) In situ labeling of DNA breaks and apoptosis by T7 DNA polymerase. Methods Mol Biol 682:37-48|