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: 8193129
• 负责人: Hans Hallen
• 金额: $ 18.66万
• 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 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8193129
• 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; Fluorescence; Fungal Genome; GC Rich Sequence; Genes; Genetic Materials; Genetic Risk; 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，我们提出了一种新的技术来监测甲基化状态，染色质状态和组蛋白修饰的基因组大小的分子，理想的整个染色体。该方法结合了遗传物质在纳米流体通道中的拉伸和近场增强下紫外共振拉曼散射的光学读出。我们预计分辨率约为1kbp或更好。该技术将为探测细胞群表观遗传多样性的单分子研究提供工具，并且应该能够产生关于肿瘤内祖细胞的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、高分辨率和无标签检测的能力，将对当前技术提供重大改进。我们实现目标的策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，我们将使用甲基化特异性荧光标记标记的DNA的纳米通道拉伸。我们还旨在展示染色质拉伸，并使用特异性抗体可视化组蛋白。在下一阶段，我们将展示与纳米流体装置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，我们将在晶圆上的测试结构上研究特定DNA和染色质结构的近场紫外共振拉曼特征。最后，我们将整合我们的发现来展示通过纳米通道扫描的DNA的近场紫外共振拉曼。此外，我们将使用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或重组酵母染色质的表观遗传图谱。我们提出了一种新的技术来监测表观遗传编程状态的基因组大小的分子，理想的整个染色体。该方法结合了遗传物质在纳米流体通道中的拉伸和近场增强下紫外共振拉曼散射的光学读出。我们期望这种方法能成为癌症研究中的一种有价值的工具。

项目成果

Fluctuation modes of nanoconfined DNA.

纳米限制 DNA 的波动模式。

• DOI: 10.1063/1.3675207
• 发表时间: 2012
• 期刊: Journal of applied physics
• 影响因子: 3.2
• 作者: Karpusenko,Alena;Carpenter,JoshuaH;Zhou,Chunda;Lim,ShuangFang;Pan,Junhan;Riehn,Robert
• 通讯作者: Riehn,Robert

Collapse of DNA under alternating electric fields.

DNA 在交变电场下的塌陷。

• DOI: 10.1103/physreve.92.012714
• 发表时间: 2015
• 期刊: Physical review. E, Statistical, nonlinear, and soft matter physics
• 作者: Zhou,Chunda;Riehn,Robert
• 通讯作者: Riehn,Robert

Chromatin modification mapping in nanochannels.

纳米通道中的染色质修饰图谱。

• DOI: 10.1063/1.4833257
• 发表时间: 2013
• 期刊: Biomicrofluidics
• 影响因子: 3.2
• 作者: Lim,ShuangFang;Karpusenko,Alena;Blumers,AnselL;Streng,DianaE;Riehn,Robert
• 通讯作者: Riehn,Robert

Collapse of DNA in ac electric fields.

• DOI: 10.1103/physrevlett.106.248103
• 发表时间: 2011-06-17
• 期刊: Physical review letters
• 影响因子: 8.6
• 作者: Zhou C;Reisner WW;Staunton RJ;Ashan A;Austin RH;Riehn R
• 通讯作者: Riehn R

Probing transient protein-mediated DNA linkages using nanoconfinement.

使用纳米限制探测瞬时蛋白质介导的 DNA 连接。

• DOI: 10.1063/1.4882775
• 发表时间: 2014
• 期刊: Biomicrofluidics
• 影响因子: 3.2
• 作者: Roushan,Maedeh;Kaur,Parminder;Karpusenko,Alena;Countryman,PrestonJ;Ortiz,CarlosP;FangLim,Shuang;Wang,Hong;Riehn,Robert
• 通讯作者: Riehn,Robert