Probes for detection of DNA accessibility in chromatin

描述（由申请方提供）：染色质中特定位点的DNA可及性变化是基因活性调节的重要决定因素。拟议的研究项目将评估能够在单分子水平上检测DNA可及性变化的新型分子探针。探针将在染色质中的特定位点组装，并将基于生物发光产生信号。我们将构建一个新的分裂荧光素酶系统，它将产生足够高的光子通量，使单分子检测。使用哺乳动物细胞培养系统和适当的DNA转染载体，我们将评估分子探针设计的性能，以监测，虽然在体内成像，在细胞经历正常的发育过程中，在基因组中的特定位点的染色质可及性的变化。评价标准将包括常规生物学实验中使用的信号/噪声、信号持续性、空间分辨率、时间分辨率和稳健性。

DESCRIPTION (provided by applicant): Changes in DNA accessibility at specific loci in chromatin are important determinants for the regulation of gene activity. The proposed research project will evaluate novel molecular probes capable of detecting changes in DNA accessibility at the single molecule level. The probes will be assembled at specific loci in chromatin, and will generate signals based on bioluminescence. We will construct a novel split luciferase system, which will generate a photon flux high enough to enable single molecule detection. Using mammalian cell culture systems and appropriate DNA transfection vectors, we will evaluate the performance of the molecular probe designs for the purpose of monitoring, though in vivo imaging, changes in chromatin accessibility of specific loci in the genome, as cells undergo normal developmental processes. Evaluation criteria will include signal/noise, signal persistence, spatial resolution, time resolution, and robustness for use in routine biological experiments.