Quantitative Real-Time DNA Repair Analysis Tools

项目摘要/摘要 DNA修复途径维持基因组的完整性，从而有助于预防癌症、疾病的发生 和衰老表型。此外，许多癌症治疗方法通过诱导基因组DNA损伤来发挥作用。因此， 辐射和遗传毒性反应对DNA修复蛋白和途径的关键要求 化疗药物使DNA修复蛋白成为改善对现有药物反应的主要靶点 抗癌养生法。开发特定的DNA修复抑制剂的关键是获得强大的， 高灵敏度的检测DNA修复能力的方法。此外，关键DNA修复途径中的缺陷或 蛋白质可能导致癌症的发生，也可能为治疗的选择性提供选择。其中许多 使用当前的“组学”技术可以检测出150个或更多DNA修复蛋白中的缺陷。然而， 存在许多只能使用诸如这里描述的那些功能分析来检测的缺陷。至 为了有效地开发这些工具，我们提出了两个具体目标：目标1将利用“逆向工程”战略 作为产生用于任何DNA修复或DNA的最佳dsDNA序列的新型发现平台的基础 结合蛋白。这种方法将允许单个DNA修复酶的DNA序列依赖性 影响探针(分子信标)优化。目标2将利用以下定义的优化共识序列 目标1中用于开发高选择性和特异性分子信标的逆向工程平台 探测器。为了提供多路复用能力，我们将针对多组荧光/猝灭剂对进行优化，并将 评估每种测试方法在96井、384井和1536井平台上的使用情况，以演示高通量应用。在……里面 目标2，我们将开发和优化三种用户就绪的实时荧光检测方法(DNA修复灯， DNA修复纯灯和DNA修复捕捉灯)能够使用纯化的DNA修复定量 蛋白质或细胞和组织裂解物。本文提出的定量分析方法将提供快速、高通量的 发现和验证DNA修复抑制剂的方法，将成为功能DNA的有价值的平台 细胞/肿瘤裂解物或组织抽吸物的修复测量和生物标记物分析。我们设想这是第一个进入 开发一个完整的DNA修复平台的一系列测试。

Project Summary / Abstract DNA repair pathways maintain the integrity of the genome and thereby help prevent the onset of cancer, disease and aging phenotypes. Further, many cancer treatments function by inducing genomic DNA damage. As such, the critical requirement for DNA repair proteins and pathways in response to radiation and genotoxic chemotherapeutics implicates DNA repair proteins as prime targets for improving response to currently available anti-cancer regimens. Essential to the development of specific DNA repair inhibitors is the availability of robust, highly sensitive assays to measure DNA repair capacity. In addition, defects in critical DNA repair pathways or proteins can predispose to cancer onset and may also provide an option for therapeutic selectivity. Many of these defects in the 150 or more DNA repair proteins can be detected using current "omics" technologies. However, there are many defects that can only be detected using functional assays such as those described herein. To effectively develop these tools, we suggest two specific aims: Aim 1 will utilize a "Reverse Engineering" strategy as the basis for a novel discovery platform yielding the optimal dsDNA sequence for any DNA repair or DNA binding protein. This approach will allow DNA sequence dependence of individual DNA repair enzymes to influence probe (Molecular Beacon) optimization. Aim 2 will exploit the optimized consensus sequence defined by the reverse engineering platform in Aim 1 for the development of highly selective and specific molecular beacon probes. To provide multiplexing capacity, we will optimize for multiple sets of fluor/quencher pairs and will evaluate each assay for use in 96-, 384- and 1536-well platforms to demonstrate high-throughput application. In Aim 2, we will develop and optimize three user ready real-time fluorescence-based assays (DNA Repair Lights, DNA Repair PureLights and DNA Repair CaptureLights) amenable to DNA Repair quantification using purified proteins or cell and tissue lysates. The quantitative assays proposed here will provide a rapid, high-throughput method for the discovery and validation of DNA repair inhibitors and will be a valuable platform for functional DNA Repair measurements and biomarker analysis of cell/tumor lysates or tissue aspirates. We envision this as first in a series of assays towards the development of a complete DNA Repairomics platform.