Novel tools to comprehensively map dynamic organization of RNA and DNA in higher-order nuclear structures within single cells

 描述(申请人提供)：控制基因调控的一个重要因素是细胞核的三维结构，它在不同的细胞状态下动态组装和调节。然而，这个核组织是如何建立的，它是如何动态地 细胞状态的变化在很大程度上是未知的。最近，一些核保留的长非编码RNA(LncRNAs)被证明对三维基因组组织的形成具有重要作用。目前尚不清楚数千个与染色质相关的lncRNA中有多少可能同样对塑造核组织具有重要作用。解决这个问题的主要挑战是，我们目前缺乏将RNA全面整合到我们对基因组组织的理解中的工具。目前用于绘制RNA-DNA相互作用的全基因组方法一次只能绘制一个RNA，不能提供有关其靶标的三维相互作用的信息，提供来自数百万个细胞的整体视图，并提供所有RNA分子的聚合图像，而不是单个分子分辨率的RNA在细胞核中的定位。在这里，我们的目标是开发一种新的方法，以实现对数千个单核内所有RNA分子的三维DNA靶标的全面单分子映射。首先，我们将开发一种新的全基因组测序方法，该方法将能够生成细胞核中所有RNA分子的三维DNA靶标的全面单分子图(目标1)。下一步，我们将开发新的微流控设备，它将能够测量数百到数千个单个细胞核中的RNA-DNA核隔室(目标2)。最后，我们将扩展这一技术，以研究RNA-DNA相互作用在细胞重新编程中的单细胞时间动力学，并通过测试几个已识别的RNA在核组织中的作用来功能验证这些映射(目标3)。这些方法将首次全面探索保留核的RNA在塑造核结构中的作用，从而克服一个重大障碍。此外，这些方法将为研究核结构提供变革性的工具--除了本提案中探讨的直接问题之外；我们预计这些工具将普遍适用于了解单细胞核结构的许多其他问题。

 DESCRIPTION (provided by applicant): An important factor in the control of gene regulation is the 3-dimensional organization of the nucleus, which is dynamically assembled and regulated in different cellular states. Yet, how this nuclear organization is established and how it dynamically changes across cell states is largely unknown. Recently, several nuclear- retained long non-coding RNAs (lncRNAs) have been shown to be important for shaping 3-dimensional genome organization. It is currently unknown how many of the thousands of chromatin-associated lncRNAs may similarly be important for shaping nuclear organization. The main challenge in addressing this question is that we currently lack the tools to comprehensively integrate RNA into our understanding of genome organization. Current genome-wide methods for mapping RNA-DNA interactions can only map a single RNA at a time, do not provide information about the 3-dimensional interaction of their targets, provide an ensemble view derived from millions of cells, and provide an aggregate picture across all RNA molecules, rather than single molecule resolution of RNA localization in the nucleus. Here, we aim to develop a novel approach to enable comprehensive single molecule mapping of the 3-dimensional DNA targets of all RNA molecules within thousands of single nuclei. First, we will a develop a novel genome-wide sequencing method that will enable the generation of comprehensive single molecule maps of the 3-dimensional DNA targets of all RNA molecules in the nucleus (Aim 1). Next, we will develop novel microfluidic devices that will enable the measurement of RNA-DNA nuclear compartments in hundreds to thousands of individual nuclei (Aim 2). Finally, we will extend this technology to study the single cell temporal dynamics of RNA-DNA interactions across cellular reprogramming and functionally validate these maps by testing the role of several identified RNAs in nuclear organization (Aim 3). These methods will overcome a major barrier by enabling, for the first time, comprehensive exploration of the role of nuclear-retained RNAs in shaping nuclear structure. Furthermore, these methods will provide transformative tools for studying nuclear structure - beyond the immediate questions explored in this proposal; we expect that these tools will be generally applicable to many additional questions for understanding nuclear structure in single cells.