Active Nanofluidics for Analysis of Chromatin and Genomic DNA Structures

描述(由申请人提供)：用于染色质和基因组DNA结构分析的活性纳米流体A。具体目的该项目将开发纳米技术，以满足在全基因组DNA和染色质结构分析中尚未满足的需求。这种能力将极大地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。该方法将使用平行的纳米通道，其横截面轮廓可以被可逆地调节为窄(纳米)或宽(微米)。可调谐通道将被加宽，以使相对较大的染色质或基因组DNA分子在其折叠状态下能够有效地负载。然后，在操作员的精确控制下，渠道将逐渐缩小。在纳米通道内同时施加电场将允许通道内染色质或DNA的受控线性化。展开的染色质或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：将测试和优化材料特性和加工方法，以构建平行的纳米通道阵列。纳米通道提供了对通道横截面轮廓、微流体流和表面化学的可重复控制。目的2.DNA线性化和稳定化：将开发协调和控制电场作用下通道横截面形状调整的机制和软件。将对直流电和脉冲场电流进行测试。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(48kb)作为初始测试。目的3.基于图像的线性化DNA分析：将开发计算机化的图像捕获和分析程序。作为一项初步的生物学测试，我们将对暴露于或不暴露于药物复制应激的培养哺乳动物细胞的线性化基因组DNA样本进行复制分叉检查。目的4.组蛋白相关DNA的分析：将开发设备内活细胞温和解离的程序。通过控制电流、温度和通道形态，将在通道内观察到染色质结构(包括核小体)的动态变化。 公共卫生相关声明：该项目将开发广泛有用的纳米技术，以满足全基因组DNA和染色质结构分析中尚未满足的重要需求。在这项提案中，纳米技术最初的具体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。尽管进行了大量的努力，但高等生物基因组中复制位点的有序激活在很大程度上仍未得到解释。这至少部分是由于协调激活大约10,000到100,000个复制位点的过程的复杂性，在这个过程中，这些位点不仅由序列决定，而且还由表观遗传因素决定。这种类型的分析在临床上很重要，因为复制错误与多种疾病有关，如沃纳综合征、塞克尔综合征、范可尼贫血和癌症。

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.