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

该申请是2014年至2018年由DFG资助的同名赠款的延伸。在第一个融资期，我们有三个主要目标：1。固体核磁共振（ssNMR）技术在RNA序列分配中的应用2. 开发利用ssNMR采集距离约束及其他相关结构参数的实验；3. RNA-蛋白复合物中RNA结构的计算。第一个筹资期的几乎所有目标都已轻易实现。我们已经开发了一种简单，易于管理的策略，使用易于产生的核苷酸型选择性标记rna和敏感的磁化转移方案，通过ssNMR获得anRNA的结构。此外，我们证明了我们的方法在大RNP复合物背景下对标记RNA的短片段的适用性。在第一个资助期的工作中，我们使用了13C检测实验；RNA典型的核磁共振严重重叠，以及3D实验需要使用核苷酸类型选择性标记来解决2D实验中的重叠的低灵敏度。对于含有长规范螺旋的RNA，这种策略可能会达到极限，因为共振重叠更为严重。因此，在RNA和RNP复合物的背景下，开发使三维光谱可行的策略对于将固态核磁共振建立为确定大RNA或作为大颗粒一部分的RNA结构的有效工具是必不可少的。继1H检测在蛋白质的ssNMR中取得巨大成功后，我们探索了将超快MAS、1H检测的固态核磁共振光谱应用于RNA的可能性。我们选择在ssNMR中进行RNA的1H检测是出于以下主要目标：1。利用13C在> - 100khz旋转速率下较长的寿命，进行高灵敏度的高维实验；2. 允许使用亚毫克量的材料和只有一个样品进行ssNMR的RNA结构测定，并进行均匀标记。第二个资助期的目的是开发一种基于1H检测和快速MAS率的实验方法，通过ssNMR来解决RNA的结构。

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.