Active Nanofluidics for Analysis of Chromatin and Genomic DNA Structures

用于染色质和基因组 DNA 结构分析的活性纳米流体

• 批准号: 7452691
• 负责人: SHUICHI TAKAYAMA
• 金额: $ 44.99万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-18 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7452691
• 关键词: Bacteriophage lambda; Biological; Biological Testing; Cell Nucleus; Cell physiology; Cells; Chemistry; Chromatin; Chromatin Structure; Computer software; DNA; DNA Structure; DNA biosynthesis; Devices; Disease; Dissociation; Epigenetic Process; Facility Construction Funding Category; Fanconi' s Anemia; Genetic; Genome; Genomics; Histones; Image; Life; Malignant Neoplasms; Mammalian Cell; Maps; Mechanics; Methods; Microfluidics; Molecular; Morphology; Nanotechnology; Nucleosomes; Organism; Phenotype; Physiologic pulse; Principal Investigator; Procedures; Process; Property; Public Health; Pulse taking; Sampling; Seckel syndrome; Seckel' s Syndrome; Shapes; Site; Stress; Stretching; Structure; Surface; Temperature; Testing; Translating; Werner Syndrome; base; comparative; computerized; electric field; epigenomics; genome-wide analysis; nanochannel; nanofluidic; nanometer; programs

DESCRIPTION (provided by applicant): Active Nanofluidics for Analysis of Chromatin and Genomic DNA Structures A. Specific Aims This project will develop nanotechnology to fill an unmet need in genome-wide analysis of DNA and chromatin structures. This capability will greatly enhance our understanding of how genetics and epigenetics translate the DNA-encoded information of the nucleus into cellular functions and phenotypes. The approach will use parallel nanochannels whose cross-sectional profiles can be reversibly regulated to be narrow (nanometers) or wide (micrometers). The tunable channels will be widened to enable efficient loading of the relatively large chromatin or genomic DNA molecules in their folded states. Then the channels will be gradually narrowed, under the precise control of the operator. Simultaneous application of an electric field within the nanochannel will allow controlled linearization of the chromatin or DNA inside the channels. The stretched out chromatin or DNA will be analyzed optically to map and observe genomic structures, such as replication forks, and epigenetic structures, as well as the distribution of nucleosomes, and organized chromatin regions. These capabilities will be used for comparative genomics and epigenomics of healthy and diseased/stressed cells. Aim 1. Construction of Tunable Nanochannel Arrays: Material properties and processing methods will be tested and optimized to construct parallel arrays of nanochannels. The nanochannels provide reproducible control of channel cross-sectional profile, microfluidic flow, and surface chemistry. Aim 2. DNA Linearization and Stabilization: Mechanisms and software will be developed to coordinate and control channel cross-sectional shape adjustments with electrical field application. Both direct current and pulsed-field current regimes will be tested. The nanochannel profile and electric fields will be optimized to allow linearization and stable molecular control using lambda bacteriophage DNA (48 kb) as an initial test. Aim 3. Image-based Analysis of Linearized DNA: Computerized image capture and analysis programs will be developed. As an initial biological test, we will examine replication forks on linearized genomic DNA samples from cultured mammalian cells exposed or not exposed to pharmacologic replication stress. Aim 4. Analysis of Histone-Associated DNA: Procedures for the gentle dissociation of live cells within the devices will be developed. Dynamic changes in chromatin structures, including nucleosomes, will be observed within the channels using controlled currents, temperatures, and channel morphologies. Public Health Relevance Statement: This project will develop broadly useful nanotechnology to fill an important unmet need in genome-wide analysis of DNA and chromatin structures. The specific initial biological application of the nanotechnology in this proposal will be to analyze genomic and epigenomic structures related to DNA replication. Despite intense efforts, the orderly activation of replication sites in genomes of higher organisms remains largely unexplained. This is due, at least in part, to the complexity of the process which orchestrates activation of an estimated 10,000 to million replication sites, where the sites are determined not only by sequence but by epigenetic factors as well. This type of analysis is important clinically because faulty replication is involved in a variety of diseases such as Werner syndrome, Seckel syndrome, Fanconi anemia and cancer.

描述（由申请人提供）：用于分析染色质和基因组 DNA 结构的活性纳米流体 A. 具体目标 该项目将开发纳米技术，以满足 DNA 和染色质结构全基因组分析中未满足的需求。这种能力将极大地增强我们对遗传学和表观遗传学如何将细胞核的 DNA 编码信息转化为细胞功能和表型的理解。该方法将使用平行纳米通道，其横截面轮廓可以可逆地调节为窄（纳米）或宽（微米）。可调通道将被加宽，以便能够有效加载处于折叠状态的相对较大的染色质或基因组 DNA 分子。然后通道将在操作员的精确控制下逐渐变窄。在纳米通道内同时施加电场将允许通道内染色质或 DNA 的受控线性化。拉伸的染色质或 DNA 将进行光学分析，以绘制和观察基因组结构，例如复制叉和表观遗传结构，以及核小体的分布和有组织的染色质区域。这些功能将用于健康和患病/应激细胞的比较基因组学和表观基因组学。目标 1. 构建可调谐纳米通道阵列：将测试和优化材料特性和加工方法，以构建并行的纳米通道阵列。纳米通道提供对通道横截面轮廓、微流体流动和表面化学的可重复控制。目标 2. DNA 线性化和稳定化：将开发机制和软件来协调和控制电场应用的通道横截面形状调整。直流电流和脉冲场电流状态都将被测试。使用 lambda 噬菌体 DNA (48 kb) 作为初始测试，将优化纳米通道轮廓和电场，以实现线性化和稳定的分子控制。目标 3. 基于图像的线性 DNA 分析：将开发计算机化图像捕获和分析程序。作为初步的生物学测试，我们将检查来自暴露或未暴露于药理学复制应激的培养哺乳动物细胞的线性基因组 DNA 样本的复制叉。目标 4. 组蛋白相关 DNA 分析：将开发装置内活细胞温和解离的程序。将使用受控电流、温度和通道形态在通道内观察染色质结构（包括核小体）的动态变化。 公共卫生相关性声明：该项目将开发广泛有用的纳米技术，以满足 DNA 和染色质结构全基因组分析中未满足的重要需求。该提案中纳米技术的具体初步生物学应用将是分析与 DNA 复制相关的基因组和表观基因组结构。尽管付出了巨大的努力，高等生物基因组中复制位点的有序激活在很大程度上仍然无法解释。这至少部分是由于协调大约 10,000 到数百万个复制位点激活的过程的复杂性，其中这些位点不仅由序列决定，还由表观遗传因素决定。这种类型的分析在临床上很重要，因为错误复制与多种疾病有关，例如沃纳综合征、塞克尔综合征、范可尼贫血和癌症。