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

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，我们提出了一种新的技术，用于监测基因组大小的分子，理想的是整个染色体的甲基化状态、染色质状态和组蛋白修饰。提出的方法结合了遗传物质在纳米流体通道中的拉伸，以及随后在近场增强下的紫外共振拉曼散射的光学读出。我们预计分辨率约为1KBP或更高。这项技术将为单分子研究探索细胞群体的表观遗传多样性提供工具，并应该能够提供关于肿瘤内祖细胞的重要性和进化的信息。这项拟议的研究本质上是探索性的，但由于其处理长分子、高分辨率和无标记检测的能力，将提供比现有技术显著的改进。我们实现目标的战略是在此过程中建立一些里程碑，这些里程碑本身就是重要的成就。在第一阶段，我们将对使用甲基化特异性荧光标记的DNA进行纳米通道拉伸。我们还旨在展示染色质的伸展，以及使用特定抗体对组蛋白的可视化。在下一阶段，我们将展示与纳米流体器件集成的金属结构对荧光的近场增强。分辨率应该提高10倍。然后，我们将研究晶片上测试结构上特定DNA和染色质结构的近场紫外共振拉曼特征。最后，我们将结合我们的发现来演示通过纳米通道扫描的DNA的近场紫外共振拉曼效应。此外，我们将使用在项目过程中被证明的最好的技术，拉曼或荧光，染色质或裸DNA，构建基因组模型DNA或酵母重组染色质的表观遗传学图谱。我们提出了一种新的技术来监测基因组大小的分子，理想情况下是整个染色体的表观遗传编程状态。提出的方法结合了遗传物质在纳米流体通道中的拉伸，以及随后在近场增强下的紫外共振拉曼散射的光学读出。我们期待这种方法成为癌症研究中的一种有价值的工具。

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.