Direct nanopore detection of modified RNA to probe structure and dynamics

项目摘要： RNA可能是细胞中用途最广的分子，因为它对几乎所有细胞的生长都有重要贡献。 过程但是我们仍然不了解RNA的许多方面，比如核苷酸， 修饰以及二级和三级结构协同工作以调节其许多功能。的 生物学的中心法则告诉我们，DNA被转录成RNA，然后被翻译成蛋白质。我们 我已经知道DNA有一个复杂的结构组织和共价修饰网络， 转录调控但RNA的修饰、剪接和结构涉及更复杂的问题 相互作用来调节蛋白质的产生。然而，如果没有合适的工具，我们无法探索这个世界。这里我们 建议利用新开发的直接RNA纳米孔测序平台直接询问 RNA，使我们能够检查修改RNA及其相互作用的动态剪接和RNA 结构纳米孔测序直接探测孔中分子的化学结构， 测量内源性RNA修饰;但我们首先需要了解原始电信号数据， 通过训练集的生成。为了探索RNA产生和剪接的动力学， 我们将利用这个工具箱来检查非规范核苷酸，例如4sU，我们作为代谢标记注射 for nascent新生RNA核糖核酸.对于结构探测，我们将采用标记未配对RNA的常规技术， 核苷酸，以允许高通量检查复杂的RNA结构。这些工具和分析 管道将给予新的见解转录调控机制，以及他们的影响， 人类疾病

Project Summary: RNA is perhaps the most versatile molecule in the cell due to its significant contribution to nearly every cellular process. But we still don’t really understand many aspects of how RNA features, such as nucleotide modifications and secondary and tertiary structure, work in concert to regulate its many functionalities. The central dogma of biology tells us that DNA is transcribed to RNA, which then is translated to protein. We already know that DNA has a complex web of structural organization and covalent modification which provides transcriptional control. But modifications, splicing, and structure of RNA involve an even more complex interplay to regulate protein production. Yet without appropriate tools, we cannot explore this world. Here we propose to leverage the newly developed direct RNA nanopore sequencing platform to directly interrogate RNA, enabling us to examine modifications to RNA and their interplay with the dynamics of splicing and RNA structure. Nanopore sequencing directly probes the chemical structure of the molecule in the pore, allowing us to measure endogenous RNA modifications; but we first need to understand the raw electrical signal data produced, through the generation of training sets. To explore the dynamics of RNA production and splicing, we will utilize this toolbox to examine non-canonical nucleotides, e.g. 4sU, that we inject as a metabolic label for nascent RNA. For structural probing, we will adapt conventional techniques which label unpaired RNA nucleotides to allow high-throughput examination of complex RNA structure. These tools and analysis pipelines will grant new insights into mechanisms of transcriptional regulation, as well as their implications for human disease.