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

RNA或核糖核酸是DNA的分子表亲，DNA是细胞的遗传蓝图，在化学成分上有着看似微小的差异。 这种差异，无论多么小，加上缺乏第二条互补链，使单个RNA分子能够折叠成像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。 目前的项目将调查的动力学和热力学与一组基本的RNA结构，从人类免疫缺陷病毒（HIV）中发现的发夹到假结和两个细菌RNA中发现的长距离对接结构，折叠和经历功能重要的结构重排从单碱基对到整个RNA分子。 将使用一种新的磁带头拉动方法来询问单个表面束缚的RNA分子，以克服长期存在的挑战，了解生物学上重要的RNA结构-动力学-功能关系-即短距离波动与长距离波动的耦合。 具体而言，将测试的假设，即形成的个别碱基对指导形成大的螺旋元件，这反过来又控制了可访问的拓扑结构作为RNA折叠。 预计这些结果将为RNA折叠的一般模型提供基础，同时也激发了不同群体的高中生和本科生参与这项研究，以追求STEM学位。RNA分子在生物聚合物中是独一无二的，因为它们将可遗传的序列信息与折叠成复杂的三维结构的能力结合在一起，在基因调控中具有重要的生物功能。 经过几十年的研究，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RNA折叠所表现出的具有挑战性的尺度范围-快速，亚毫秒的碱基对波动引起分钟慢，大规模的构象重排。 目前的项目为RNA研究引入了一个新的实验平台，能够询问与RNA（非）折叠相关的整个时间和长度机制。 具体而言，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拴系的RNA分子进行数小时的牵引，使用0至50 pN的宽生理相关力范围。 该项目将重点关注三种长期研究且结构复杂性不断增加的基因调节RNA：HIV TAR发夹、小preQ 1核糖开关假结和四向连接Mn 2+核糖开关。 结合计算建模，这组目标预计将有助于揭示序列和配体结合RNA（非）折叠的贡献，在前所未有的细节。 作为补充，将采取多管齐下的方法，让传统上代表性不足的高中生和本科生参与研究，旨在对附近的底特律市产生影响。 因此，该项目旨在利用技术来展示教育研究活动，让更广泛的公众参与到一个因COVID-19大流行而成为焦点的科学主题- RNA中。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.