Semi-artificial nanomachines for detection of DNA damage and apoptosis

描述(由申请人提供)：多功能分子大小的机器系统的开发将是未来生物和医学技术的基石。该项目的目标是通过将分子马达蛋白质的活性生物分子与人工工程组件相结合，开发出第一个实用的纳米机器设计。该结构将是一台分子大小的机器，属于我们最近推出的一类新的半人工纳米设备。它将通过荧光标记物的共价连接对特定的DNA断裂进行超快标记。自组装后，机器将使用自己的材料制造两个探测器单元。在存在DNase I型(含5‘PO4)或DNase II型(含5’OH)的钝端DNA断裂时，探测器单元将选择性地连接到其目标DNA末端，并用红色或绿色荧光团标记它们。所描述的结构将是第一个检测固定组织切片中两种特定DNA断裂的纳米传感器，以及一个在几秒钟内检测活的非固定细胞中DNA损伤和凋亡的超高速传感器。该项目的具体目标是：1.设计和测试一种半人工纳米机器，能够超快地检测溶解DNA中不同类型的DNA损伤。2.发展基于DNA酶I和II类DNA断裂标记的新型纳米机器作为纳米传感器的生物医学应用，以检测固定组织中两种主要类型的细胞凋亡。3.开发新设计的纳米机器作为超快速纳米传感器在生物医学中的应用，以检测未固定的活细胞中的DNA损伤和凋亡。在Aim1中，我们将使用溶液中特定数量的DNA断裂的模型系统来测试检测的选择性、特异性和速度。在目标2中，我们将把我们的新构造物应用于固定的组织切片，并将其作为原位检测细胞凋亡的纳米传感器的应用。在目标3中，我们将开发我们的构建物作为一种超快传感器的应用，在没有固定细胞的情况下检测活细胞的凋亡。这种半人工纳米机器将展示一种新的仿生方法来设计超快分子尺寸的设备，并将成为一种独特的工具，用于检测生物和医学研究中的DNA损伤和细胞凋亡。

DESCRIPTION (provided by applicant): 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.