IIBR Multidisciplinary: Exact internuclear distance and dynamics measurements in RNA molecules by a novel nuclear magnetic resonance technique

科罗拉多大学安舒茨医学中心获得了一项奖励，以开发一种核磁共振（NMR）协议，该协议可以常规地确定高分辨率核糖核酸（RNA）结构及其动力学，仅基于经验数据和适度的实验努力。虽然将核磁共振数据转换为原子间距离需要对基础物理和数学有深入的了解，但待开发的软件将使这些知识变得不必要。该项目特别关注RNA，但其中一些方法也将提高对蛋白质的适用性。结合预期的测量时间减少，这将使该方案对核磁共振波谱和结构生物学社区具有吸引力。在教育方面，RNA分子的结构动力学研究在很大程度上没有得到代表，而科学界的焦点集中在平均结构代表上。大分子及其相互作用本质上是动态的，这就是为什么学习如何评估与结构并行的运动是至关重要的。因此，指导学生如何弥合这一差距是本项目的关键部分，特别是考虑到近年来大分子动力学实验的一般领域发展迅速。将招收一名来自科罗拉多大学RNA生物科学计划暑期实习项目的暑期学生，该项目为来自研究项目有限的机构的学生提供获得顶级研究经验的机会。RNA不仅是将遗传密码翻译成蛋白质的模板，而且还具有多种重要的细胞功能。理解这些功能完全依赖于对原子分辨率结构排列的了解，以及越来越明显的RNA分子的构象动力学。几乎一半已确定的RNA结构已被核磁共振解解。然而，仅从最流行和最成功的核磁共振探针——核检波器增强（NOE），很少能获得高分辨率的RNA结构。相反，需要许多额外的半经验限制和只有专家才能获得的劳动密集型技术来获得结构平均值，并且只有少数实验推导的结构集合代表现实的空间采样。因此，结构生物学界需要新的方法来改善可收集和用于RNA结构测定的结构数据池。原则上，NOE直接取决于两个原子之间的距离。然而，NOE被用作半定量的上限距离约束。这种约束的不精确性质意味着关于结构和动力学的重要信息丢失了。我们的想法是精确测量NOE (eNOE)，它可以转换成一个严格的距离限制。在理想情况下，这种距离的测量精度可以达到10-11米，并且可以对RNA分子中的数百个质子对进行测量。这些项目建议建立一个有效的协议，使用精确的NOE （eNOE）方法来改善各种大小RNA的核磁共振结构，使RNA研究人员能够计算小RNA的多态结构集合，并改善较大RNA的平均结构或特定局部结构方面。该项目的智力优势在于，eNOE距离将改善所有类型的核磁共振结构的确定：i)小rna（最多20个核苷酸）的结构可以在没有任何其他限制的情况下以高分辨率定义；eNOE还可以用于计算多态结构集合，以真实地采样其构象空间，ii)更大的RNA分子将导致改善的平均结构。我们将提供核磁共振脉冲序列代码和我们的eNOE分析程序eNORA免费下载从我们的网页。该方案将有助于研究人员以更高的分辨率研究RNA结构，这是更好地了解RNA功能的先决条件。该奖项反映了美国国家科学基金会的法定使命，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。

An award is made to the University of Colorado Anschutz Medical Center to develop a Nuclear Magnetic Resonance (NMR) protocol to routinely determine high-resolution ribonucleic acid (RNA) structures and their dynamics based exclusively on empirical data with modest experimental effort. Although the conversion of the NMR data into interatomic distances requires in-depth understanding of the underlying physics and mathematics, the software to be developed will render this knowledge unnecessary. The project specifically concerns RNA, but some of the methods will boost the applicability to proteins as well. In combination with the anticipated reduction in measuring time, this will make the protocol attractive to the NMR spectroscopy and structural biology communities. Educationally, structural dynamics studies of RNA molecules have been largely unrepresented while the scientific communities' focus has centered on average structural representation. Macromolecules and their interactions are dynamic in nature and this is why it is critical to learn how to evaluate motions in parallel with structure. Thus, mentoring students on how to bridge this gap is a critical part of this proposed project, especially considering that the general field of macromolecular dynamics experimentation has moved quickly within recent years. A summer student from the RNA Bioscience Initiative \ Summer Internship Program at the University of Colorado will be recruited, which offers access to top-level research experience for students from institutions with limited research programs.RNA not only is the template for translating the genetic code into proteins, but also carries out diverse important cellular functions. Understanding these functions absolutely depends on knowledge of the structural arrangement at atomic resolution, and, as is becoming increasingly evident, the conformational dynamics of RNA molecules. Almost one-half of the determined RNA structures have been solved by NMR. However, high-resolution RNA structures can rarely be obtained from the most popular and successful NMR probe alone, the Nuclear Overhauser Enhancement (NOE). Instead, many additional semi-empirical restraints and labor-intensive techniques only accessible to experts are required to obtain a structural average, and there are only a few experimentally derived ensembles of structures representing realistic spatial sampling. Therefore, the structural biology community is in need of novel methods that improve the pool of structural data that can be collected and used for RNA structure determination. In principle, the NOE directly depends on the distance between two atoms. However, the NOE is employed as a semi-quantitative upper limit distance restraint. The non-exact nature of this restraint means that important information about structure and dynamics is lost. It is our idea to measure the NOE exactly (eNOE), which can be converted into a tight distance limit. In ideal cases, such a distance can be measured to an accuracy of ca. 10-11 meters and can be obtained for hundreds of proton pairs in an RNA molecule. These project proposes to establish an efficient protocol to improve NMR structures of RNA of all sizes using the exact NOE (eNOE) approach, enabling RNA researchers to calculate multi-state structural ensembles for small RNAs, and improving average structures or specific local structural aspects for larger RNAs. It is the intellectual merit of this project that the eNOE distance will improve all types of determined NMR structures: i) structures of small RNAs (up to 20 nucleotides) may be defined at high resolution without any other restraints; the eNOE can also be used to calculate multi-state structural ensembles to realistically sample their conformational space, ii) larger RNA molecules will result in improved average structures. We will offer NMR pulse sequence codes and our eNOE analysis program eNORA for free download from our webpage. This protocol should help researchers to study RNA structures at higher resolution, a prerequisite for better understanding of RNA function.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.