DNA Repair-on-a-Chip: Spatially Encoded Microwell Arrays

描述(申请人提供)：为了抑制癌症、神经变性、衰老和其他疾病，保持基因组的完整性是必不可少的。与基因组保存不同的是DNA损伤，它可能导致突变、序列重排和细胞毒性。DNA损伤是不可避免的，因为DNA损伤剂存在于我们的环境和细胞中。为了抵消DNA损伤的有害影响，我们进化出了复杂的DNA修复系统。现在已经知道，每一条主要的DNA修复途径都能抑制癌症。此外，由于癌症通常使用DNA损伤剂治疗，因此肿瘤的DNA修复能力调节对化疗的敏感性也就不足为奇了。尽管DNA损伤和修复的测量很重要，但它远远不是常规的，主要是因为缺乏可靠和快速的DNA损伤检测。在这里，通过汇聚工程师、生物学家和计算机程序员的专业知识，我们建议通过开发一个快速半自动单细胞DNA损伤量化平台来满足这一需求，该平台可以广泛分布，并易于被公共卫生研究人员应用， 学术界、工业界和医学界。正如第一阶段提交的文件所定义的那样，我们创建并测试了96孔CometChip平台的原型，并优化了工程设计和生产设备，以生产空间编码的20孔和96孔Comet凝胶。充分证明，添加含有细胞外基质蛋白(EMPs)的微孔Comet凝胶支持人类细胞生长长达两周，EMPs不影响彗星的形成。为了能够确定商业使用的化学品、环境中发现的化学品或新开发的药物的遗传毒性，并量化DNA修复能力，而不需要确定特定的DNA修复技术。这项旨在开发“DNA芯片修复”技术的提案结合了基于琼脂糖基的微孔阵列、空间编码细胞识别、自动化数据处理和细胞外基质蛋白质的使用，以优化、验证和商业化一系列空间编码微孔阵列。我们将展示我们已显著改进了制造工艺(Aim1)，开发了可生产96井和384井CometChips的巨型井成型机(目标2)，并提议实施用于数据分析的图形用户界面(目标3)。最后，我们将通过分析NTP文库中一系列化合物对DNA损伤和修复反应的影响来严格验证这项新技术，并揭示DNA损伤反应的个体间和细胞间类型差异(目标4)。通过整合生物学和工程学中的传统方法，这里描述的芯片平台上的DNA修复代表着一项重大的技术进步，提供了高通量、客观和定量的测量，有可能成为DNA损伤分析的新标准。

DESCRIPTION (provided by applicant): Preserving genomic integrity is essential in order to suppress cancer, neurodegeneration, aging and other diseases. At odds with genomic preservation is DNA damage, which can drive mutations, sequence rearrangements and cellular toxicity. DNA damage is unavoidable, as DNA damaging agents are present in our environment and in our cells. To counteract the deleterious effects of DNA damage, we have evolved sophisticated DNA repair systems. It is now known that every major DNA repair pathway suppresses cancer. Furthermore, since cancer is often treated using DNA damaging agents, it is not surprising that the DNA repair capacity of tumors modulates sensitivity to chemotherapy. Despite its importance, measurements of DNA damage and repair are far from routine, primarily due to the lack of reliable and rapid DNA damage assays. Here, by bringing together convergent expertise among engineers, biologists and computer programmers, we propose to meet this need by developing a platform for rapid semi- automated single-cell DNA damage quantification that can be broadly distributed and readily applied by researchers in public health, academia, industry and medicine. As defined in the Phase I submission, we created and tested a prototype for a 96-well CometChip platform and have optimized the engineering design and a production apparatus to produce spatially encoded 20 and 96 well demonstrated that supplementation of the Microwell Comet gels with extracellular matrix proteins (EMPs) supports the growth of human cells for up to two weeks and the EMPs do not impact the formation of comets. To enable characterization of the genotoxicity of chemicals used commercially, those found in the environment or newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair technology. This proposal, to develop the 'DNA Repair on a Chip' technology, combines the use of agarose based Microwell arrays, spatially encoded cellular recognition, automated data processing, and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays. We will demonstrate that we have significantly advanced the manufacturing process (Aim1), have developed a macrowell former to produce 96-well and 384-welll CometChips (Aim 2), and propose the implementation of a graphical user interface for data analysis (Aim 3). Finally, we will rigorously validate this new technology by analyzing the genotoxic effects of a range of compounds from the NTP library for their impact on DNA damage and repair responses and to reveal inter-individual and inter-cell type variation in DNA damage responses (Aim 4). Through the integration of traditional methods in biology and engineering, the DNA Repair on a Chip platform described here represents a significant technological advance, providing high-throughput, objective, and quantitative measurements that have the potential to become a new standard in DNA damage analysis.