EPIGENETIC PROFILING BY NEAR-FIELD UV-RAMAN SCATTERING IN NANOCHANNELS


Year of Award:
2009
Award Type:
R21
Project Number:
CA132075
RFA Number:
RFA-CA-08-006
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
RIEHN, ROBERT
Other PI or Project Leader:
HALLEN, HANS
Institution:
NORTH CAROLINA STATE UNIVERSITY RALEIGH
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.