A New High-Throughput Technology To Reveal The Dynamic Functional States of RNAs

描述(由申请人提供)：后基因组时代已经迎来了对RNA在整个细胞中所起的广泛和核心作用的深刻的新的认识。我们现在面临的挑战是阐明RNA的潜在功能机制，以真正理解、设计和纠正它们在生物系统和疾病中的功能。然而，由于在细胞的动态、非平衡环境中询问RNA结构的技术挑战，我们对RNA在生命最基本过程中所扮演角色的基本序列-结构-功能关系的理解仍处于起步阶段。由于缺乏高通量工具来表征‘组学’规模的RNA结构，这一点变得更加混乱。因此，这项提议的科学目标是通过开发一种能够确定整个基因组中RNA的动态功能状态的“组学”技术来应对这两个挑战。最近，随着高通量RNA结构表征技术的发展，我们在这个方向上迈出了突破性的一步。这项技术结合了RNA结构化学探测和下一代测序，在体外同时探测数百个RNA的结构。在这里，我们试图将这一技术扩展到表征RNA的动态、共转录的折叠途径，并阐明RNA的功能在多大程度上取决于它在活跃转录时经历的折叠途径。我们发现RNA分子动态折叠路径的创新方法颠覆了这个问题：我们不是监测单个RNA分子随时间的折叠过程，而是拍摄整个RNA分子群体的快照，并以统计方式重建它们的折叠轨迹。因此，我们的创新技术创造性地结合了下一代测序的吞吐量和灵敏度、化学RNA结构探测的多功能性以及统计学的力量，首次创造了一种新的方法来阐明RNA结构及其折叠路径中形成的相互作用。这项技术将在提出基本问题的背景下开发，这些问题包括平衡RNA折叠和共转录RNA折叠之间的差异，配体在转录过程中如何与RNA相互作用并指导其折叠路径，以及新生RNA折叠如何耦合到甚至调节转录动力学。我们预计这项工作的结果将是我们思考RNA结构-功能关系的方式的转变，从而为我们理解RNA分子如何在生命最基本的过程中发挥无处不在的、多功能的和关键的作用创造了一个新的范式。

DESCRIPTION (provided by applicant): The post-genomic era has ushered in a profound new appreciation of the broad and central roles played by RNAs across the cell. We now face the challenge of elucidating the underlying functional mechanisms of RNAs to truly understand, engineer, and correct their function in biological systems and disease. However, our understanding of the fundamental sequence-structure-function relationship underlying RNA's role in life's most basic processes is still in its infancy because of the technical challenge of interrogating RNA structures in the dynamic, non-equilibrium environment of the cell. This is further confounded by a lack of high throughput tools that can characterize RNA structures on an 'omics' scale. Therefore, the scientific objective of this proposal is to address both of these challenges by developing an 'omics' technology that can determine the dynamic functional states of RNAs across the genome. We recently made a breakthrough step in this direction with our development of a high-throughput RNA structure characterization technology. This technology combines RNA structure chemical probing and next-generation sequencing to probe the structures of hundreds of RNAs simultaneously in vitro. Here, we seek to extend this technology to characterize the dynamic, co-transcriptional folding pathways of RNAs, and elucidate the extent to which an RNA's function is determined by the folding pathway it undergoes as it is actively transcribed. Our innovative approach to uncovering the dynamic folding pathways of RNA molecules turns the problem on its head: rather than monitor the folding processes of individual RNA molecules over time, we instead take snapshots of entire populations of RNA molecules and statistically reconstruct their folding trajectories. Our innovative technology is thus a creative combination of the throughput and sensitivity of next- generation sequencing, the versatility of chemical RNA structure probing, and the power of statistics to create a new approach to elucidate RNA structures and interactions formed during their folding pathways for the first time. This technology will be developed in the context of asking fundamental questions about the differences between equilibrium and co-transcriptional RNA folds, how ligands interact with RNAs during transcription and guide their folding pathways, and how nascent RNA folding couples to and even regulates transcription dynamics. We anticipate the outcome of this work will be a transformation in the way we think about the RNA structure-function relationship, thereby creating a new paradigm in our understanding of how RNA molecules perform ubiquitous, versatile and critical roles in life's most fundamental processes.