Epigenetic profiling by near-field UV-Raman scattering in nanochannels

通过纳米通道中的近场紫外拉曼散射进行表观遗传分析

• 批准号: 7694949
• 负责人: Hans Hallen
• 金额: $ 23.38万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7694949
• 关键词: Achievement; Antibodies; Arts; Base Composition; Cells; Chromatin; Chromatin Modeling; Chromosomes; Cytosine; DNA; DNA Binding; Data; Data Quality; Detection; Development; Devices; Dyes; Environmental Risk Factor; Epigenetic Process; Evolution; Figs - dietary; Fluorescence; Fungal Genome; GC Rich Sequence; Genes; Genetic; Genetic Materials; Genomics; Goals; Haploidy; Haplotypes; Histones; Human Genome; Hybrids; Imagery; Individual; Label; Libraries; Location; Malignant Neoplasms; Maps; Measures; Metals; Methods; Methylation; Modeling; Modification; Molecular Analysis; Monitor; Nature; Neoplasm Metastasis; Optics; Population; Process; Property; Research; Resolution; Scanning; Signal Transduction; Staging; Stem cells; Stretching; Structure; Surface; Techniques; Technology; Testing; Ursidae Family; Yeasts; anticancer research; cancer stem cell; chromatin modification; driving force; genome sequencing; genome-wide; histone modification; improved; nanochannel; nanofluidic; performance tests; programs; reconstitution; single molecule; tool; tumor

DESCRIPTION (provided by applicant): Epigenetic programming has emerged as a central theme in cancer development. Here we propose a new technique for monitoring the methylation state, chromatin status, and histone modification of genomic sized molecules, ideally whole chromosomes. The proposed method combines stretching of genetic material in nanofluidic channels, and subsequent optical readout by UV-resonant Raman scattering under near-field enhancement. We expect a resolution of about 1 kbp or better. The technique will provide a tool for single-molecule studies probing the epigenetic diversity of cell populations, and should be able to yield information about the importance and evolution of progenitor cell within tumors. The proposed research is exploratory in nature, but will provide significant improvements over the current art because of its capability to handle long molecules, high resolution, and label-free detection. Our strategy toward our goal is to establish a number of milestones along the way, which by themselves are significant achievements. In the first stage, we will nanochannel stretching of DNA that is labeled using methylation-specific fluorescent markers. We also aim at demonstrating chromatin stretching, and visualization of histones using specific antibodies. In the next stage, we will demonstrate that near-field enhancement of fluorescence by metal structures that are integrated with the nanofluidic device. The resolution should improve by a factor of 10. We will then investigate the near-field UV-resonant Raman signatures of specific DNA and chromatin constructs on test structures on wafers. Finally, we will integrate our findings to demonstrate near-field UV-resonant Raman of DNA scanned through nanochannels. Further, we will use the best technology proven in the course of the project, Raman or fluorescence, chromatin or bare DNA, to construct an epigenetic map of genomic model DNA or reconstituted chromatin from yeasts. We propose a new technique for monitoring the epigenetic programming state of genomic sized molecules, ideally whole chromosomes. The proposed method combines stretching of genetic material in nanofluidic channels, and subsequent optical readout by UV-resonant Raman scattering under near-field enhancement. We expect the method to become a valuable tool in cancer research.

描述（由申请人提供）：表观遗传编程已成为癌症发展的中心主题。在这里，我们提出了一种新技术，用于监测基因组大小分子的甲基化状态，染色质状态和组蛋白的修饰，理想的整个染色体。所提出的方法结合了纳米流体通道中遗传物质的拉伸，以及随后在近场增强下进行的紫外线谐音拉曼散射的光学读数。我们预计分辨率约为1 kbp或更高。该技术将为探测细胞群体表观遗传多样性的单分子研究提供工具，并应能够产生有关肿瘤中祖细胞的重要性和演变的信息。拟议的研究本质上是探索性的，但由于其处理长分子，高分辨率和无标签检测的能力，因此将对当前的艺术进行重大改进。我们针对我们目标的战略是在此过程中建立许多里程碑，这本身就是重大成就。在第一阶段，我们将使用甲基化特异性荧光标记标记的DNA的纳米渠道拉伸。我们还旨在证明染色质拉伸和使用特定抗体的组蛋白可视化。在下一阶段，我们将证明通过与纳米流体设备集成的金属结构对荧光的近场增强。分辨率应提高10倍。然后，我们将研究特异性DNA和染色质构建体的近场紫外线拉曼特征，并在晶片上测试结构上进行染色质构建体。最后，我们将整合我们的发现，以展示通过纳米通道扫描的DNA的近场紫外线拉曼。此外，我们将使用在项目过程中证明的最佳技术，拉曼或荧光，染色质或裸露的DNA，构建基因组模型DNA的表观遗传图或酵母重构染色质的表观遗传图。我们提出了一种新技术，用于监测基因组大小分子的表观遗传编程状态，理想的整个染色体。提出的方法结合了纳米流体通道中遗传物质的拉伸，以及随后在近场增强下进行紫外线谐振拉曼散射的光学读数。我们希望该方法成为癌症研究中的宝贵工具。