MINIATURIZED INSTRUMENTATION FOR THE HTS OF DNA REPAIR

描述(由申请人提供)：修复DNA损伤的能力对细胞的生存至关重要，无论是正常细胞还是癌症细胞。DNA损伤后，DNA修复基因和其他因子被激活，以消除损伤，或者，如果DNA损伤太大，就会启动细胞程序性死亡。目前的大多数抗癌化疗药物依赖于它们产生不可修复的DNA损伤，导致细胞凋亡/细胞死亡的能力。大量证据表明，核苷酸切除修复(NER)是化疗药物耐药的重要来源。顺铂-DNA加合物被NER去除，这是已知的从DNA中去除铂-DNA链内加合物的唯一机制。尽管NER机制很复杂(目前已在该途径中发现了20种蛋白质)，但NER机制可以概括为五个基本步骤：识别DNA损伤；切割受损的链和损伤；切除受损的链，产生缺口；合成新的DNA来填补缺口；以及连接最终的缺口。研究表明，当NER抑制剂与顺铂联合应用时，抑制NER将为增强顺铂的细胞毒性和克服顺铂耐药性提供一种合理的途径。在这项建议中，我们描述了一种有效、灵敏、可靠但具有成本效益的分析和仪器，用于筛选大量化合物以确定可商业化的NER抑制剂。人们花费了大量的精力来寻找“简单”的DNA NER单罐分析方法，但没有一种方法被证明具有足够的选择性、精确度和实用性，能够为这一最重要的细胞功能提供可靠的高通量分析。我们认为，唯一实用和可靠的检测NER的方法将需要使用高分辨率凝胶电泳法，这种方法将单链DNA片段分离到碱基分辨。我们已经发起了与理查·马蒂斯教授的合作。大学化学系。帮助设计和实施毛细管电泳(CE)设备，该设备具有HTS用于DNA修复抑制剂所需的所有特性。该系统将使用：微制毛细管阵列电泳板，最终将设计为每个反应通道接受少于2微升的反应体积；微板本身内置的DNA分离程序；以及与为微制阵列设计的荧光检测系统兼容的新型DNA底物。由于荧光DNA底物的成本相对较高，以及筛选过程中所需的无细胞提取物和克隆酶分离物，因此需要小体积的检测。使用小体积系统的小型化仪器提供了主要的成本优势。在第一阶段，我们将确定拟议仪器的可行性和设计特点。第二阶段将侧重于可制造性和对大约10,000种天然产品的成功筛选，以验证新型CE系统。

DESCRIPTION (provided by applicant): The ability to repair DNA damage is critical to the survival of a cell, whether normal or cancerous. Upon damage, DNA repair genes and other factors are activated either to remove the damage, or, if the DNA damage is too extensive, to initiate programmed cell death. The majority of current anticancer chemotherapies rely on their ability to create unrepairable DNA lesions, leading to apoptosis/cell death. A large body of evidence proves that Nucleotide Excision Repair (NER) is an important source of resistance to chemotherapeutic reagents. Cisplatin-DNA adducts are removed by NER, the only mechanism known by which platinum-DNA intrastrand adducts are removed from DNA. Although complex (>20 proteins have so far been identified in the pathway), NER mechanisms can be summarized by five basic steps: recognition of the DNA lesion; cleavage of the damaged strand and of the lesion; excision of the damaged strand, creating a gap; synthesis of new DNA to fill the gap; and ligation of the final nick. Studies indicate that inhibition of NER would provide a rational approach to enhancing cisplatin's cytotoxicity and overcoming cisplatin resistance when the NER inhibitor is administered in combination with cisplatin. In this proposal we describe an effective, sensitive, reliable yet cost-effective assay and instrumentation for use in screening large numbers of compounds to identify commercializable inhibitors of NER. Substantial effort has been expended in search of "easy," single pot assays for DNA NER, but none has proven to be sufficiently selective, precise, and practical to lead to a reliable high throughput assay for this most important of cellular function. We believe that the only practical and reliable assay for NER will require the use of a high resolution gel electrophoresis which separates single stranded DNA fragments to basepair resolution. We have initiated a collaboration with Prof. Richard Mathies, Dept. of Chemistry, Univ. of California, Berkeley, to help with the design and implementation of a Capillary Electrophoresis (CE) apparatus that has all the properties required for HTS for DNA repair inhibitors. The system will use: microfabricated capillary array electrophoresis microplates, which will ultimately be designed to accept reaction volumes of less than 2 microliters per reaction channel; a DNA isolation procedure built into the microplate itself; and novel DNA substrates which are compatible with the fluorescence detection system designed for the microfabricated arrays. The need for small volumes for this assay arises from the relatively high cost of fluorescent DNA substrates required for assay success and the Cell-Free Extracts and cloned enzyme isolates required during the screening. Miniaturized instrumentation using a small volume system provides a major cost advantage. During Phase I, we will establish the feasibility and design characteristics of the proposed instrumentation. Phase 2 will focus on manufacturability and the successful screening of approximately 10,000 natural products as validation of the novel CE system.