Quantitative Real-Time DNA Repair Analysis Tools

定量实时 DNA 修复分析工具

• 批准号: 8646260
• 负责人: Jay George
• 金额: $ 20.21万
• 依托单位: TREVIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-11 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8646260
• 关键词: 3-methyladenine-DNA glycosylase; Address; Aging; Alkylating Agents; Antibodies; Aspirate substance; Base Excision Repairs; Biological Assay; Biological Markers; Cells; Consensus; Consensus Sequence; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Repair Pathway; DNA Sequence; DNA glycosylase; DNA repair protein; DNA-Binding Proteins; Defect; Dependence; Development; Disease; Engineering; Enzymes; Fluorescence; Funding; Genome; Genomic DNA; Genomics; Goals; Grant; Human; Individual; Lesion; Malignant Neoplasms; Measurement; Measures; Methods; Methylation; MicroRNAs; Modification; Molecular; Molecular Probes; Mutation; Oligonucleotides; Pathway interactions; Phase; Phenotype; Proteins; Radiation; Recombinant Proteins; Regimen; Research Personnel; Seeds; Series; Structure; Technology; Therapeutic; Time; Tissues; Tumor Tissue; University of Pittsburgh Cancer Institute; Validation; Veins; base; cancer cell; cancer therapy; design; endonuclease; improved; inhibitor/antagonist; mRNA Expression; mRNA Stability; neoplastic cell; new technology; novel; novel strategies; prevent; promoter; protein expression; public health relevance; repaired; research clinical testing; response; temozolomide; tool; tumor

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.

项目总结/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 和老化表型。此外，许多癌症治疗通过诱导基因组DNA损伤起作用。因此，在本发明中， DNA修复蛋白和途径对辐射和遗传毒性反应的关键要求 化疗药物涉及DNA修复蛋白作为改善对目前可用的 抗癌疗法开发特异性DNA修复抑制剂的关键是获得稳定的， 高灵敏度的检测来测量DNA修复能力。此外，关键DNA修复途径或 蛋白质可以使癌症发病易感，并且还可以提供治疗选择性的选择。许多这些 150种或更多的DNA修复蛋白中的缺陷可以使用当前的“组学”技术检测。但是，在这方面， 有许多缺陷只能使用功能测定法（例如本文所述的那些）来检测。到 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 作为一个新的发现平台的基础，为任何DNA修复或DNA修复提供最佳dsDNA序列。 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。Aim 2将利用由以下定义的优化的共有序列： Aim 1中的逆向工程平台，用于开发高选择性和特异性的分子信标 probes.为了提供多路复用能力，我们将针对多组荧光剂/猝灭剂对进行优化，并将 评估每种测定法在96孔、384孔和1536孔平台中的使用，以证明高通量应用。在 目标2，我们将开发和优化三个用户准备实时荧光检测（DNA修复灯， DNA Repair PureLights和DNA Repair CaptureLights）适合于使用纯化的DNA Repair定量。 蛋白质或细胞和组织裂解物。本文提出的定量分析将提供一种快速、高通量的方法， 该方法用于发现和验证DNA修复抑制剂，并将成为功能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志物分析。我们设想这是第一次在 一系列试验，以开发一个完整的DNA修复平台。