Unveiling Functionally Critical, Ephemeral RNA (un)folding States with Magnetic Tape Head Tweezers

使用磁带头镊子揭示功能关键的短暂 RNA（解）折叠状态

• 批准号: 2140320
• 负责人: Nils Walter
• 金额: $ 67.5万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2140320&HistoricalAwards=false
• 关键词: Unveiling; Functionally; Critical; Ephemeral; RNA

RNA, or ribonucleic acid, is the molecular cousin of DNA, the genetic blueprint of the cell, and bears a seemingly minor difference in chemical composition. This difference, however small, together with the absence of a second, complementary strand, enables single RNA molecules to fold into structures that can be as intricate as those of proteins. This folding lays the foundation for a multitude of cellular RNA functions, particularly controlling and executing gene expression, including of viruses and bacteria. The current project will investigate the kinetics and thermodynamics with which a foundational set of RNA structures, ranging from a hairpin found in the human immunodeficiency virus (HIV) to a pseudoknot and a long-range docking architecture found in two bacterial RNAs, fold and undergo functionally important structural rearrangements from single base pairs to the entire RNA molecule. Bridging the associated broad time and length scales will be achieved using a novel magnetic tape head pulling approach to interrogate individual surface tethered RNA molecules, with the goal of conquering a long-standing challenge in understanding biologically important RNA structure-dynamics-function relationships― that of the coupling of short- with long-range fluctuations. Specifically, the hypothesis will be tested that the formation of individual base pairs guides the formation of large helical elements, which in turn govern the accessible topology as the RNA folds. It is anticipated that the results will provide the basis for general models of RNA folding while also inspiring a diverse group of high school and undergraduate students getting involved in this research to pursue a STEM degree.RNA molecules are unique among biopolymers in that they couple inheritable sequence information with the ability to fold into complex three-dimensional structures with important biological functions in gene regulation. After decades of study, the precise links of RNA sequence with folding and function are only starting to emerge, in part due to the challenging range of scales exhibited by RNA folding – fast, sub-millisecond base pair fluctuations give rise to minute-slow, large-scale conformational rearrangements. The current project introduces a novel experimental platform for RNA studies, capable of interrogating this entire time and length regime relevant to RNA (un)folding. Specifically, preliminary data demonstrate the use of a magnetic tape head force spectrometer to pull for hours at microsecond time resolution on single superparamagnetic-bead tethered RNA molecules, using a wide, physiologically relevant force range of 0 to 50 pN. This project will focus on three long-studied gene regulatory RNAs of increasing structural complexity: the HIV TAR hairpin, the small preQ1 riboswitch pseudoknot, and the four-way junction Mn2+ riboswitch. Combined with computational modeling, this set of targets is anticipated to help reveal the contributions of sequence and ligand binding to RNA (un)folding in unprecedented detail. Complementarily, a multi-pronged approach will be pursued to involve traditionally underrepresented high school and undergraduate students in research, aiming for an impact in the nearby city of Detroit. This project thus aims to leverage technology to present educational research activities that engage the broader public in a scientific topic – RNA – cast into the spotlight by the COVID-19 pandemic.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

RNA（即核糖核酸）是 DNA 的分子表亲，是细胞的遗传蓝图，并且在化学成分上具有看似微小的差异。这种差异，无论多么小，再加上缺乏第二条互补链，使得单一 RNA 成为可能。分子折叠成与蛋白质一样复杂的结构，这种折叠为多种细胞RNA功能奠定了基础，特别是控制和执行基因表达，包括病毒和细菌的基因表达。当前的项目将研究动力学和热力学。以此为基础一组 RNA 结构，从人类免疫缺陷病毒 (HIV) 中发现的发夹到两个细菌 RNA 中发现的假结和长程对接结构，折叠并经历从单碱基对到整个 RNA 分子的功能上重要的结构重排桥接相关的广泛时间和长度尺度将通过使用一种新颖的磁带头牵引方法来询问单个表面束缚的RNA分子来实现，目标是克服理解生物学上重要的RNA的长期挑战具体来说，将检验以下假设：单个碱基对的形成引导大螺旋元件的形成，而大螺旋元件又控制可访问的拓扑结构。预计这些结果将为 RNA 折叠的一般模型提供基础，同时也激励不同的高中生和本科生参与这项研究，攻读 STEM 学位。RNA 分子在生物聚合物中是独一无二的。他们夫妇经过数十年的研究，RNA序列与折叠和功能的精确联系才刚刚开始出现，部分原因是其范围具有挑战性。 RNA 折叠所表现出的尺度——快速、亚毫秒级的碱基对波动会引起分钟级缓慢、大规模的构象重排。当前的项目引入了一种用于 RNA 研究的新型实验平台，能够探究整个时间和长度机制的相关性。到RNA具体来说，初步数据表明，使用磁带头力谱仪以微秒时间分辨率对单个超顺磁珠系留的 RNA 分子进行数小时的拉伸，使用 0 至 50 pN 的广泛生理相关力范围。该项目将重点关注三个长期研究的结构复杂性不断增加的基因调控RNA：HIV TAR 发夹、小 preQ1 核糖开关假结和四路连接 Mn2+与计算模型相结合，这组目标预计将有助于以前所未有的细节揭示序列和配体结合对 RNA（解）折叠的贡献，作为补充，将采取多管齐下的方法来涉及传统上代表性不足的高中和大学。因此，该项目旨在利用技术开展教育研究活动，让更广泛的公众参与因 COVID-19 大流行而成为人们关注的科学主题——RNA。 .这个奖项体现了通过使用基金会的智力价值和更广泛的影响审查标准进行评估，NSF 的法定使命被认为值得支持。