Decoding chromosome structure with multiplexed super-resolution microscopy

摘要 几十年的研究表明，基因组组织是非随机的，并对许多核产生至关重要的影响 包括转录调控、DNA复制和DNA修复在内的过程，以及越来越多的证据 这表明染色体采用的三维结构对发育至关重要，而且 经常受到疾病的困扰。我们目前的许多认识都来自于我们所使用的生化技术 在大量的细胞群体上，导致我们在理解建立和 维护组织状态，特别是在单个单元的上下文中。我们建议引进一套新的 基于单细胞技术的单分子超分辨率成像方法DNA-Paint to 通过一套同时具有高多解性和空间分辨率的工具来弥合这一差距。具体而言，在 目的1我们将开发一种多路(20色)超分辨率染色体成像策略来成像 基因组目标的长度从千碱基到数兆碱基，这将使我们能够研究 染色质纤维在单个细胞中在一定长度范围内的折叠特性。在目标2中，我们将开发 多重分析以共定位单个细胞中的蛋白质、RNA分子和特定的基因组位置 纳米级的。然后，我们将在大染色质的模型中心研究结构蛋白的组织 人类不活跃的X染色体上的环路。在目标3中，我们将开发一种与距离相关的超分辨率 一种探测蛋白质和DNA靶标之间的特定相互作用的方法，将允许敏感的 检测拥挤环境中的分子相互作用。我们将部署这项技术来查询 单个细胞中上述染色质环状中心的组成和表观遗传状态。总而言之， 我们的方法将提出许多关于特定基因的定位、组成和表观遗传状态的问题 研究人员首次能够接触到个体细胞中的基因组位置，并有望影响不同的领域 超越了染色体生物学。

Summary Decades of study have revealed that genome organization is non-random and critically impacts many nuclear processes including the regulation of transcription, DNA replication, and DNA repair, and increasing evidence suggests that the three-dimensional structures adopted by chromosomes are critical for development and are often perturbed in disease. Much of our current understanding comes from biochemical techniques performed on large populations of cells, leading to many gaps in our understanding of the mechanisms that establish and maintain organizational states, particularly in the context of individual cells. We propose to introduce a new set of single-cell technologies based on the single-molecule super-resolution imaging method DNA-PAINT to bridge this gap with a suite of tools possessing both high multiplexibility and spatial resolution. Specifically, in Aim 1 we will develop a multiplexed (>20 color) super-resolution chromosomal imaging strategy to image genomic targets ranging from kilobases to multiple megabases in Iength, which will enable us to investigate the folding properties of the chromatin fiber in single cells over a range of length-scales. In Aim 2, we will develop multiplexed assays to co-localize proteins, RNA molecules, and specific genomic sites in individual cells at the nanoscale. We will then investigate organization of architectural proteins at a model hub of large chromatin loops on the human inactive X-chromosome. In Aim 3, we will develop a proximity-dependent super-resolution method to probe specific interactions between protein and DNA targets that will allow for the sensitive detection of molecular interactions in crowded environments. We will deploy this technology to query the composition and epigenetic states of the aforementioned chromatin looping hub in individual cells. Collectively, our methods will make many questions about the positioning, composition, and epigenetic states of specific genomic loci in individual cells accessible to researchers for the first time, and promise to impact diverse fields beyond chromosome biology.