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: 7614542
• 负责人: SHUICHI TAKAYAMA
• 金额: $ 39.46万
• 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/7614542
• 关键词: Bacteriophage lambda; Biological; Biological Testing; Cell Nucleus; Cell physiology; Cells; Cellular Stress; Chemistry; Chromatin; Chromatin Structure; Computer software; DNA; DNA Structure; DNA biosynthesis; Devices; Disease; Dissociation; Epigenetic Process; 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; 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 association study; genome-wide analysis; nanochannel; nanofluidic; nanometer; programs; public health relevance

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.具体目的该项目将开发纳米技术，以填补对DNA和染色质结构的全基因组分析的未满足需求。这种能力将大大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。该方法将使用平行的纳米通道，其横截面轮廓可以可逆地调节为窄（纳米）或宽（微米）。可调通道将扩大，以便在其折叠状态下有效地加载相对较大的染色质或基因组DNA分子。然后，在操作员的精确控制下，通道将逐渐缩小。纳米通道内的电场同时应用将允许控制通道内的染色质或DNA的线性化。伸展的染色质或DNA将在光学上分析，以绘制和观察基因组结构，例如复制叉和表观遗传结构，以及核小体的分布以及有组织的染色质区域。这些能力将用于健康和患病/胁迫细胞的比较基因组学和表观基因组学。 AIM 1。可调纳米通道阵列的构造：将测试并优化材料属性和处理方法，以构建纳米渠道的平行阵列。纳米通道可重复控制通道横截面剖面，微流体流和表面化学。 AIM 2。DNA线性化和稳定：将开发机制和软件，以协调和控制通道横截面形状与电场应用。将测试直流电流和脉冲场电流状态。纳米通道轮廓和电场将被优化，以使用lambda噬菌体DNA（48 kb）作为初始测试，允许线性化和稳定的分子对照。 AIM 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。作为初始生物学测试，我们将检查来自暴露于药物复制应力或未暴露于药物复制应力的培养哺乳动物细胞的线性基因组DNA样品的复制叉。目标4。对组蛋白相关的DNA的分析：将开发设备内活细胞轻轻解离的程序。将使用受控电流，温度和通道形态观察到染色质结构（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，以满足对DNA和染色质结构的全基因组分析的重要未满足需求。该提案中纳米技术的特定初始生物学应用将是分析与DNA复制有关的基因组和表观基因组结构。尽管做出了巨大的努力，但在较高生物体的基因组中的复制位点有序地激活仍无法解释。这至少部分归因于该过程的复杂性，该过程策划了估计的10,000到百万复制位点的激活，在该过程中，这些位点不仅由序列而是通过表观遗传因素确定。这种类型的分析在临床上很重要，因为有缺陷的复制涉及多种疾病，例如Werner综合征，Seckel综合征，Fanconi贫血和癌症。