Mapping epigenetic modifications at the nanoscale: Aptamers for microscopy

描述（由申请人提供）：我们建议开发一种通过单分子直接成像来绘制染色质中组蛋白翻译后化学修饰的技术。我们的方法是为原子力显微镜（AFM）使用一种新的成像模式，该模式允许直接读取化学成分，同时获得高分辨率的分子图像。通过这种方式，我们将观察到翻译后修饰的模式，如乙酰化，以及随之而来的染色质结构的局部修饰。该模式要求同源配体连接到AFM探针上。抗体尚未被证明足以满足这一目的，因此我们建议开发具有以下特点的合成配体：(a)高可重复性(b)化学简单稳定(c)能够识别小的化学修饰。具体而言，我们建议：1。开发对乙酰化组蛋白具有高度特异性的DNA适体。2. 使用麻省大学医学院Peterson实验室开发的人工乙酰化组蛋白H4阵列，在AFM的同步地形和化学成像模式（“识别成像”）下测试新的适配体。在马歇尔大学乔治实验室对从小鼠细胞系中提取的启动子染色质进行初步识别成像研究。这项提议的工作将为一个更雄心勃勃的项目铺平道路，该项目旨在利用原子力显微镜对极小样本的要求，从一个（或几个）细胞中分析染色质。

DESCRIPTION (provided by applicant): We propose to develop a technique for mapping post-translational chemical modifications of histones in chromatin by direct imaging of single molecules. Our approach is to use a new imaging mode for the atomic force microscope (AFM) that allows chemical composition to be read directly and at the same time as a high resolution molecular image is acquired. In this way, we will observe both the pattern of post-translational modifications, such as acetylation, and the consequent local modifications of chromatin structure. The mode requires that a cognate ligand be tethered to the AFM probe. Antibodies have not proved adequate for this purpose, so we propose to develop synthetic ligands that are (a) highly reproducible (b) chemically simple and stable and (c) capable of recognizing small chemical modifications. Specifically, we propose to: 1. Develop DNA aptamers that are highly specific for acetylated histones. 2. Test the new aptamers in the simultaneous topography and chemical imaging mode of the AFM ('recognition imaging') using artificial acetylated histone H4 arrays developed in the Peterson lab at the University of Massachusetts Medical School. 3. Carry out an initial recognition imaging study of promoter chromatin extracted from a mouse cell line at the Georgel lab at Marshall University. The proposed work will pave the way for a more ambitious project aimed at analyzing chromatin from one (or a few cells) by exploiting the very small sample requirements of atomic force microscopy.