LC-ESI-Mass Spectrometer for the Analysis of Highly-Modified RNA- and DNA-Derivatives

法兰克福歌德大学自然科学的研究重点之一是RNA的研究-从转录的调节，其随后的加工（例如剪接）一直到翻译的调节。特别令人感兴趣的是不编码蛋白质的RNA。过去15年的特点是知识的巨大增长，特别是在后一领域，冠状病毒大流行表明RNA具有很大的治疗潜力。重要的是能够合成新的RNA衍生物，例如具有增加的稳定性或新的功能。这些功能可以是智能探针，以收集关于RNA的新知识，或者特别是用于调节RNA功能的新机制。例如，开发以光作为外部触发剂调节RNA活性的方法是法兰克福开发的专业之一。该研究目前由例如SFB 902（基于RNA的调节的分子原理）或GRK 1986（复杂光控制）资助。对于所有这些项目，需要分析新合成的寡核苷酸衍生物。表征的金标准是质谱法-这是无可替代的。同样通过本申请人的贡献，现在可以引入一系列新的修饰，并且新的化学酶促固相支持的合成策略使得能够合成比以往更长和更高修饰的衍生物。它们的分析非常复杂，仅为此目的需要质谱仪。在过去，在同一设备上分析其他物质类别也伴随着严重的问题。目前可用的器械正在接近其最大使用寿命。对于新设备，需要降低检测限并提高分辨率。还特别要求新装置现在还能够执行片段化和片段分析，并且因此能够提供序列分析。前者是重要的，因为对于更长和更复杂的探针，到目前为止，很大一部分储备用于表征。后者将允许更好地分析衍生物，特别是在寡核苷酸在合成后用需要优化的反应进一步衍生化的情况下。

One of the research focusses of the natural sciences at Goethe University Frankfurt is the investigation of RNA - ranging from the regulation of transcription, its subsequent processing (for example splicing) all the way to the regulation of translation. Of particular interest is RNA that does not code for proteins. The past 15 years are characterized by an enormous increase of knowledge especially in the latter field and the corona pandemic has shown that RNA has a large therapeutic potential. It is essential to be able to synthesize new RNA derivatives for example with increased stability or new functionalities. Such functionalities can be intelligent probes to gather new knowledge about RNA or especially also new mechanisms for the regulation of the RNA's function. For example, the development of methods for the regulation of RNA activity with light as external trigger is one of the specialities developed in Frankfurt. This research is currently funded for example by SFB 902 (Molecular Principles of RNA-Based Regulation) or GRK 1986 (Complex Light Control).For all of these projects the newly synthesized oligonucleotide derivatives need to be analyzed. The gold standard for the characterization is mass spectrometry - and this is without alternative. Also by contributions of the present applicants, an array of new modifications can now be introduced and new chemoenzymatic, solid-phase supported synthesis strategies enable the synthesis of longer and higher modified derivatives than ever before. Their analysis is very complicated and requires a mass spectrometer only for this purpose. Also analyzing other substance classes on the same device was accompanied with severe problems in the past. The currently available device is approaching its maximal life time. With a new device the detection limit needs to be lowered and the resolution needs to be enhanced. It is especially also required that the new device is now also able to perform fragmentation and fragment analysis and can hence provide sequence analysis. The former is important since with longer and more complex probes a significant part of the stock was used up for the characterization up to now. The latter will allow better analysis of the derivatives, especially in cases where the oligonucleotide is further derivatized after synthesis with reactions that need to be optimized.