Detection and Identification of new RNA modifications

该项目的中心目标是发现和鉴定迄今未知的RNA修饰。检测本身集中在液相色谱和质谱（LC-MS）和稳定同位素标记技术的组合上。中性丢失筛选法（NLS）是一种用于检测所有含核糖核苷的方法，已从大肠杆菌的tRNA制备物中鉴定出100多种候选化合物。杆菌其中37个高质量的数据集表明，它们的结构可以在短时间内得到阐明。作为概念验证，研究了一个候选者的结构，后来命名为msms 2 i6 A。我们可以证明，这种新的修饰含有硫缩醛结构，它是由自由基SAM酶MiaB对i6 A的重复作用产生的。在第二个资助期内，我们建议澄清更多候选分子的结构，并在来自不同来源的RNA制剂中检测新的候选信号。后者可能导致估计现有RNA结构的新“上限”数量。此外，将通过在已知修饰酶的敲除菌株中筛选它们的存在来研究不同候选物的生物发生。我们还希望通过分析它们在生物功能组装体（如多聚核糖体）中的存在，更深入地了解各种候选物的生物相关性。

The central objective of this project is the discovery and identification of hitherto unknown RNA modifications. The detection itself is centered on a combination of liquid chromatography and mass spectrometry (LC-MS) and stable isotope labelling techniques. A so called neutral loss screening (NLS) method, developed for the general detection of all ribose-containing nucleosides, has identified >100 candidate compounds in tRNA preparations from E. coli . For 37 of these high-quality data sets suggest that their structures can be elucidated in short order. As a proof-of-concept, the structure of one candidate, later named msms2i6A, was investigated. We could show that this new modification contains a thioacetal structure, which is generated by the repeated action of the radical-SAM enzyme MiaB on i6A. For the second grant period, we propose to clarify the structures of several more candidates, and to detect new candidate signals in RNA preparations from various origins. The latter may lead to the estimation of a new “ceiling” number of existing RNA structures. Furthermore, the biogenesis of different candidates will be investigated by screening their presence in knockout strains of known modification enzymes. We also expect to gain more insight into the biological relevance of various candidates by analyzing their presence in biologically functional assemblies such as polysomes.