Fluorescent Molecular Oscillator for DNA Damage Detection

描述（由申请人提供）：在本提案中，我们将设计和测试一种新的和有用的纳米器件-荧光分子振荡器。我们将开发一个应用程序，该设备作为一个超快的纳米传感器检测DNA损伤和细胞悬浮液中的凋亡。该传感器是一类半人工分子机器的第一个代表，这是我们最近引入的一类分子器件。在我们的纳米器件中，分子马达蛋白连续分离并重新连接两个不同的荧光标记的DNA部分，这两个部分参与荧光共振能量转移（FRET）。当细胞中存在特定的DNA断裂时，传感器各部分之间的能量传递中断，导致荧光颜色变化，从而检测DNA损伤和凋亡。检测反应非常快，不到一分钟。该项目的具体目标是：1。为了设计和测试这种新型的分子装置-发光分子振荡器，它将自组装从荧光标记的DNA和分子马达蛋白-牛痘拓扑异构酶I。2.开发新创建的荧光分子振荡器作为基于FRET的纳米传感器的第一个实际生物医学应用，用于超快速检测活细胞和固定细胞中的特定DNA损伤和凋亡。在实验中，我们将确定传感器的灵敏度、特异性和足以准确检测DNA断裂的反应时间。还将测量分子振荡器在反应条件下的存活时间。基于我们的研究结果，我们将修改原型设计，并将其应用于超快速检测固定和活细胞悬浮液中的凋亡DNA断裂。该纳米传感器将在细胞凋亡和药物开发的理论和临床研究中，以及在其中细胞凋亡和DNA损伤的程度具有预后价值的疾病的治疗效果的评估中是有用的。拟议的项目将导致开发一种新的工具，以满足医疗诊断和病理学的需要。该技术将简化并使其更便宜地评估细胞死亡和DNA损伤程度具有预后价值的疾病的治疗效果，如阿尔茨海默病和各种癌症。

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.