Semi-artificial nanomachines for detection of DNA damage and apoptosis

描述（由申请人提供）：多功能分子大小机器系统的开发将是未来生物学和医学技术的基石。该项目的目标是通过将分子马达蛋白的活性生物分子与人工工程组件相结合来开发第一个实用的纳米机器设计。该结构将是一个分子大小的机器，属于我们最近推出的一类新的半人工纳米器件。它将通过共价连接荧光标记物来执行特异性DNA断裂的超快速标记。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DNA酶I型（带有5'PO 4）或DNA酶II型（带有5' OH）的平端DNA断裂时，检测器单元将选择性地附着到它们的靶DNA末端，并将用红色或绿色荧光团标记它们。所描述的构建体将是第一个检测固定组织切片中两种类型的特异性DNA断裂的纳米传感器，以及在几秒钟内检测活的非固定细胞中的DNA损伤和凋亡的超快速传感器。该项目的具体目标是：1。设计和测试一种半人工纳米机器，能够超快速检测溶解DNA中不同类型的DNA损伤。2.开发新设计的纳米机器的生物医学应用，将其用作基于DNase I和II型DNA断裂的标记的用于检测固定组织中两种主要类型的细胞凋亡的纳米传感器。3.开发新设计的纳米机器的生物医学应用，将其用作超快速纳米传感器，用于检测非固定活细胞中的DNA损伤和凋亡。在Aim 1中，我们将使用溶液中特定量DNA断裂的模型系统来测试选择性，特异性和检测速度。在目标2中，我们将把我们的新结构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。在目标3中，我们将开发我们的构建体作为超快速传感器的应用，在不固定的情况下检测活细胞中的凋亡。这种半人工纳米机器将为设计超高速分子尺寸的器件提供一种新的仿生方法，并将成为生物和医学研究中检测DNA损伤和凋亡的独特工具。

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.