Detection and Identification of new RNA modifications

该项目的中心目标是发现和鉴定迄今为止未知的RNA修饰。检测本身是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。一种所谓的中性损失筛选（NLS）方法，用于所有含核糖核苷的一般检测，已经从大肠杆菌的tRNA制剂中鉴定出了100个候选化合物。这些高质量的数据集中有37个表明它们的结构可以在短时间内被阐明。作为概念验证，研究了一个候选的结构，后来被命名为msms2i6A。我们可以证明这种新的修饰含有硫缩醛结构，这是由自由基- sam酶MiaB对i6A的重复作用产生的。对于第二个资助期，我们建议澄清更多候选信号的结构，并在不同来源的RNA制备中检测新的候选信号。后者可能导致对现有RNA结构的新“上限”数量的估计。此外，不同的候选生物发生将通过筛选它们在已知修饰酶的敲除菌株中的存在来研究。我们也希望通过分析它们在多聚体等生物功能组装中的存在，来深入了解各种候选物的生物学相关性。

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.