Active Nanofluidics for Analysis of Chromatin and Genomic DNA Structures

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.具体目标本项目将开发纳米技术，以填补DNA和染色质结构全基因组分析的未满足需求。这种能力将大大提高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，其横截面可以可逆地调节为窄（纳米）或宽（微米）。可调通道将被加宽，以使相对较大的染色质或基因组DNA分子在其折叠状态下有效加载。然后，在操作员的精确控制下，通道将逐渐缩小。在纳米通道内同时施加电场将允许通道内染色质或DNA的可控线性化。延伸的染色质或DNA将被光学分析，以绘制和观察基因组结构，如复制叉，表观遗传结构，以及核小体的分布，和有组织的染色质区域。这些能力将用于健康和患病/应激细胞的比较基因组学和表观基因组学。目的1。可调谐纳米通道阵列的构建：将测试和优化材料性能和加工方法，以构建纳米通道并行阵列。纳米通道提供了通道横截面、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：将开发机制和软件来协调和控制电场应用下的通道横截面形状调整。直流电和脉冲场电流都将被测试。纳米通道轮廓和电场将被优化，以允许线性化和稳定的分子控制，使用lambda噬菌体DNA （48 kb）作为初始测试。目标3。基于图像的线性化DNA分析：计算机图像捕获和分析程序将被开发。作为最初的生物学测试，我们将检查线性化基因组DNA样本的复制叉从培养的哺乳动物细胞暴露或不暴露于药物复制压力。目标4。组蛋白相关DNA分析：将开发设备内活细胞温和解离的程序。染色质结构（包括核小体）的动态变化将通过控制电流、温度和通道形态在通道内观察到。

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.