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

全面绘制单细胞内高阶核结构中 RNA 和 DNA 动态组织的新工具

• 批准号: 9144436
• 负责人: Mitchell Guttman
• 金额: $ 70.2万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9144436
• 关键词: 3-Dimensional; Address; Cell Count; Cell Nucleus; Cells; Chromatin; Communities; Complex; DNA; Equipment; Gene Expression Regulation; Generations; Genome; Goals; Health; Individual; Knowledge; Lead; Maps; Measurement; Measures; Methods; Microfluidic Analytical Techniques; Microfluidic Microchips; Microfluidics; Microscopy; Nuclear; Nuclear Structure; Play; Probability; RNA; Resolution; Role; Shapes; Sorting - Cell Movement; System; Technology; Testing; Time; Training; Untranslated RNA; crosslink; genome-wide; human disease; novel; novel strategies; single molecule; tool

 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.

 描述（由申请人提供）：控制基因调控的一个重要因素是细胞核的三维组织，其在不同的细胞状态下动态组装和调控。然而，这个核组织是如何建立的， 细胞状态之间的变化在很大程度上是未知的。最近，几种核保留的长非编码RNA（lncRNA）已被证明对于形成三维基因组组织是重要的。目前尚不清楚成千上万的染色质相关lncRNA中有多少可能对塑造核组织同样重要。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单个RNA，不能提供关于其靶标的三维相互作用的信息，提供来自数百万细胞的整体视图，并提供所有RNA分子的聚合图，而不是RNA在细胞核中定位的单分子分辨率。在这里，我们的目标是开发一种新的方法，使全面的单分子映射的三维DNA靶的所有RNA分子在数千个单个细胞核。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谱（Aim 1）。接下来，我们将开发新型微流体设备，能够测量数百到数千个细胞核中的RNA-DNA核区室（目标2）。最后，我们将扩展这项技术来研究跨细胞重编程的RNA-DNA相互作用的单细胞时间动力学，并通过测试几种已鉴定的RNA在核组织中的作用来功能性地验证这些图谱（Aim 3）。这些方法将克服一个主要障碍，使第一次，全面探索核保留的RNA在塑造核结构的作用。此外，这些方法将为研究核结构提供变革性工具-超越本提案中探索的直接问题;我们预计这些工具将普遍适用于理解单细胞核结构的许多其他问题。