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，用于监测甲基化状态，染色质状态，和组蛋白修饰的基因组大小的分子，理想的是整个染色体。所提出的方法结合了纳米流体通道中的遗传物质的拉伸，以及随后通过近场增强下的UV共振拉曼散射的光学读出。我们希望分辨率约为1 kbp或更高。该技术将为探索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内祖细胞重要性和进化的信息。拟议的研究本质上是探索性的，但由于其处理长分子、高分辨率和无标记检测的能力，将提供对现有技术的重大改进。我们实现目标的战略是在这条道路上沿着建立一些里程碑，这些里程碑本身就是重大成就。在第一阶段，我们将使用甲基化特异性荧光标记物标记的DNA的纳米通道拉伸。我们的目标也是展示染色质的拉伸，以及使用特异性抗体的组蛋白的可视化。在下一阶段，我们将证明与纳米流体装置集成的金属结构的近场荧光增强。分辨率应提高10倍。然后，我们将研究近场紫外共振拉曼签名的特定DNA和染色质结构上的测试结构的晶片。最后，我们将整合我们的研究结果，以证明近场紫外共振拉曼的DNA扫描通过纳米通道。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DNA，构建基因组模型DNA或来自酵母的重组染色质的表观遗传图谱。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表观遗传编程状态。所提出的方法结合了纳米流体通道中的遗传物质的拉伸，以及随后通过近场增强下的UV共振拉曼散射的光学读出。我们希望这种方法能成为癌症研究中一种有价值的工具。

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.