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在整个细胞中发挥的广泛和核心作用的深刻的新认识。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，工程和纠正它们在生物系统和疾病中的功能。然而，我们对RNA在生命最基本过程中的作用的基本序列-结构-功能关系的理解仍然处于起步阶段，因为在细胞的动态非平衡环境中询问RNA结构的技术挑战。这进一步被缺乏可以在“组学”规模上表征RNA结构的高通量工具所混淆。因此，该提案的科学目标是通过开发一种可以确定整个基因组中RNA的动态功能状态的“组学”技术来解决这两个挑战。我们最近在这方面取得了突破性进展，开发了高通量RNA结构表征技术。该技术结合了RNA结构化学探测和下一代测序，可在体外同时探测数百种RNA的结构。在这里，我们试图扩展这种技术来表征RNA的动态共转录折叠途径，并阐明RNA的功能在多大程度上取决于它被积极转录时所经历的折叠途径。我们的创新方法揭示了RNA分子的动态折叠途径，将问题转化为问题：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，并统计重建其折叠轨迹。因此，我们的创新技术是下一代测序的通量和灵敏度，化学RNA结构探测的多功能性和统计学的力量的创造性组合，以创造一种新的方法来阐明RNA结构和相互作用形成的折叠途径的第一次。这项技术将在询问有关平衡和共转录RNA折叠之间的差异，配体如何在转录过程中与RNA相互作用并指导其折叠途径，以及新生RNA折叠如何耦合甚至调节转录动力学的基本问题的背景下开发。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.