Development of versatile molecular-size machine systems will be the cornerstone of future technologies in biology and medicine. The goal of this project is to develop the first practical nanomachine design by integrating an active biological molecule of a molecular motor protein with artificially engineered components. The construct will be a molecular-size machine belonging to a new class of semi-artificial nanodevices, which we recently introduced. It will perform super-fast labeling of specific DNA breaks via covalent attachment of fluorescent markers. After self-assembly, the machine will use its own material to fabricate two detector units. In the presence of blunt-ended DNA breaks of DNase I type (bearing 5' PO4) or DNase II type (bearing 5' OH), the detector units will selectively attach to their target DNA ends and will label them with either red or green fluorophores. The described construct will be the first nanosensor detecting two types of specific DNA breaks in fixed tissue sections and a super-fast sensor detecting DNA damage and apoptosis in live non-fixed cells within seconds.
Specific Aims of the project are: 1. To design and test a semi-artificial nanomachine capable of super-fast detection of different types of DNA damage in solubilized DNA. 2. To develop a biomedical application of the newly designed nanomachine employing it as a nanosensor for detection of two major types of apoptosis in fixed tissues, based on labeling of DNase I and II type DNA breaks. 3. To develop a biomedical application of the newly designed nanomachine employing it as a super-fast nanosensor for detection of DNA damage and apoptosis in non-fixed live cells.
In Aim1 we will use model systems with specific amounts of DNA breaks in solution to test selectivity, specificity and speed of detection.
In Aim 2 we will apply our new construct to fixed tissue sections and will develop its application as a nanosensor for in situ detection of apoptosis.
In Aim 3 we will develop an application of our construct as a super-fast sensor detecting apoptosis in live cells without their fixation. The semi-artificial nanomachine will exemplify a new bionic approach to the design of super-fast molecular-size devices and will be a unique tool for detection of DNA damage and apoptosis useful in biological and medical research. ? ? ?
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