Fluorescent Molecular Oscillator for DNA Damage Detection

DESCRIPTION (provided by applicant): In this proposal, we will design and test a new and useful nanodevice - a fluorescent molecular oscillator. We will develop an application for this device as an ultra-fast nanosensor for detection of DNA damage and apoptosis in cell suspensions. The sensor is the first representative of a class of semi-artificial molecular machines, a category of molecular devices which we recently introduced. In our nanodevice, the molecular motor protein continuously separates and religates two different fluorescently labeled DNA parts, which participate in fluorescence resonance energy transfer (FRET). When specific DNA breaks are present in cells, energy transfer between parts of the sensor is interrupted, leading to a fluorescence color change signaling detection of DNA damage and apoptosis. The detection reaction is very fast and takes less than a minute. Specific Aims of the project are: 1. To design and test this new type of molecular device - a light-emitting molecular oscillator, which will self-assemble from fluorescently labeled DNA and a molecular motor protein - vaccinia topoisomerase I. 2. To develop the first practical biomedical application of the newly created fluorescent molecular oscillator as a FRET-based nanosensor for the ultra-fast detection of specific DNA damage and apoptosis in live and fixed cells. In experiments we will determine sensitivity, specificity of the sensors and the time of reaction sufficient for the accurate detection of DNA breaks. The time of survival of molecular oscillators in reaction conditions will also be measured. Based on our results, we will modify the prototypical design and apply it for ultra-fast detection of apoptotic DNA breaks in suspensions of fixed and live cells. The nanosensor will be useful in theoretical and clinical research in apoptosis and drug development, and in evaluation of the effects of therapy in diseases in which the extent of apoptosis and DNA damage have prognostic value. The proposed project will result in the development of a new tool for the needs of medical diagnostics and pathology. The technology will simplify and make it cheaper evaluation of the effects of therapy in diseases in which the extent of cell death and DNA damage have prognostic value, such as Alzheimer's disease, and various cancers.