Development of solid-state NMR methodology to study RNA and protein-RNA complexes

开发用于研究 RNA 和蛋白质-RNA 复合物的固态 NMR 方法

• 批准号: 424767449
• 负责人: Professorin Dr. Teresa Carlomagno
• 金额: --
• 依托单位: School of Biosciences
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/424767449?language=en
• 关键词: Development; solid; state; NMR; methodology

This application is the extension of a grant with the same name, which has been funded by the DFG from 2014 to 2018. In the first funding period, we had three main goals: 1. Development of solid-state NMR (ssNMR) experiments for the sequential assignment of nucleotides in RNA; 2. Development of experiments to collect distance restraints and other relevant structural parameters by ssNMR; 3. Calculation of the RNA structure in an RNA-protein complex. Nearly all goals of the first funding period have been readily achieved. We have developed a straightforward, manageable strategy that uses easy-to-produce nucleotide-type selective-labeled RNAs and sensitive magnetization transfer schemes to obtain the structure of anRNA by ssNMR. In addition, we proved the applicability of our method to short stretches of labelled RNA in the context of large RNP complexes.In the work conducted in the first funding period, we used 13C detected experiments; the severe overlap of the NMR resonances typical for RNA and the low sensitivity of 3D experiments required using nucleotide type selective labeling to resolve overlaps in 2D experiments. This strategy is likely to reach its limits for RNA containing long canonical helices, where resonance overlap is more severe. Thus, the development of strategies that make 3–dimensional spectra feasible in the context of RNA and RNP complexes is indispensable to establish solid-state NMR as a valid tool for structural determination of either large RNAs or RNA as part of large particles. Following the great success of 1H detection in ssNMR of proteins, we explored the possibility of applying ultrafast MAS, 1H-detected solid-state NMR spectroscopy to RNA. Our choice to move towards 1H detection for RNA in ssNMR is motivated by to main goals: 1. Take advantage of the much longer life time of 13C at >100 kHz spinning rates to perform high-dimensionality experiments with high sensitivity; 2. Allow structural determination of RNA by ssNMR using submilligram quantities of material and only one sample with uniform labeling. The aim of this second funding period is to develop an experimental approach, based on 1H detection and fast MAS rates, to solve the structure of RNA by ssNMR.

该申请是DFG从2014年至2018年资助的同名赠款的延伸。在第一次融资期间，我们有三个主要目标：1。发展固态核磁共振（ssNMR）实验，用于RNA中核苷酸的顺序分配; 2.通过ssNMR收集距离约束和其他相关结构参数的实验开发; 3.计算RNA-蛋白质复合物中的RNA结构。第一个供资期的几乎所有目标都已顺利实现。我们已经开发了一种简单的，可管理的策略，使用易于生产的核苷酸类型的选择性标记的RNA和敏感的磁化转移计划，以获得的ssNMR的anRNA的结构。此外，我们证明了我们的方法的适用性，在大的RNP complex.In的背景下，在第一个资助期进行的工作中，我们使用13 C检测实验的标记RNA的短延伸;严重重叠的NMR共振典型的RNA和低灵敏度的3D实验需要使用核苷酸类型选择性标记，以解决重叠在2D实验。对于含有长规范螺旋的RNA，这种策略可能达到其极限，其中共振重叠更严重。因此，发展战略，使三维光谱可行的背景下的RNA和RNP复合物是必不可少的，以建立固态NMR作为一个有效的工具，无论是大RNA或RNA的大颗粒的一部分结构测定。在蛋白质ssNMR中1H检测的巨大成功之后，我们探索了将超快MAS，1H检测的固态NMR光谱应用于RNA的可能性。我们选择在ssNMR中对RNA进行1H检测是出于以下主要目标：1.利用13 C在>100 kHz旋转速率下更长的寿命，以高灵敏度进行高维实验; 2.允许通过ssNMR使用亚毫克量的材料和仅一个具有均匀标记的样品来确定RNA的结构。第二个资助期的目的是开发一种基于1H检测和快速MAS速率的实验方法，通过ssNMR解决RNA的结构。