Metabolic labeling of RNA by means of synthetic and bioorthogonally reactive nucleoside as fluorogenic probes in living cells

通过合成和生物正交反应核苷作为活细胞中的荧光探针对 RNA 进行代谢标记

• 批准号: 461961063
• 负责人: Professor Dr. Hans-Achim Wagenknecht
• 金额: --
• 依托单位: Institut für Organische Chemie (IOC)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/461961063?language=en
• 关键词: Metabolic; labeling; RNA; means; synthetic

In order to investigate biological processes of nucleic acids, in particular RNA, in their natural environment, inside living cells, they have to be conjugated with fluorescent labels. The metabolic labeling has been established for RNA. However, in nearly all published examples the metabolic labeling of RNA and DNA was –independently of the applied reaction type- performed with fixed cells, and not in living cells. In this project, the metabolic labeling of RNA will be further developed by means of synthetic and bioorthogonally reactive nucleosides to a fluorogenic tool. By combination with FISH probes, RNA transcript will be not only visualized as snapshots in fixed cells but also in living cells. The metabolically labeled RNA works as fluorogenic energy donor, the FISH probes as energy acceptor. The labeling applies Diels-Alder reactions with inverse electron demand and “photoclick” reactions, because they were successfully approved for metabolic labeling in living cells, and thus are the organic-chemical basis for this project. Cyclopropenes will be used as bioorthogonally reactive groups, because they are only slightly bigger than vinyl groups, they are both sufficiently reactive and sufficiently stable functional groups, they can be synthesized in a straightforward way, and thus, they are suitable for metabolic labeling. 1-Methylcyclopropenes will be used for metabolic Diels-Alder reactions and 3-methylcyclopropenes for metabolic “photoclick” reactions. In vitro experiments characterize their reactivity and kinetics. The metabolic labeling will be analyzed in HeLa cells by RNA hydrolysis. The fluorogenic properties is evaluated in combination with FISH probes on selected mRNA transcripts and miRNA sequences.

为了研究核酸，特别是RNA，在其自然环境，活细胞内的生物过程，它们必须与荧光标记结合。RNA的代谢标记已经建立。然而，在几乎所有已发表的例子中，RNA和DNA的代谢标记——与应用的反应类型无关——都是在固定细胞中进行的，而不是在活细胞中进行的。在本项目中，RNA的代谢标记将通过合成和生物正交反应核苷的荧光工具进一步发展。通过与FISH探针的结合，RNA转录不仅可以在固定细胞中以快照的形式可视化，而且可以在活细胞中可视化。代谢标记的RNA作为荧光能量供体，FISH探针作为能量受体。标记应用Diels-Alder反应与逆电子需求和“光点击”反应，因为它们已成功批准用于活细胞的代谢标记，因此是本项目的有机化学基础。环丙烯将被用作生物正交反应基团，因为它们只比乙烯基稍大，它们都是足够活跃和足够稳定的官能团，它们可以用简单的方法合成，因此，它们适合用于代谢标记。1-甲基环丙烯将用于代谢性Diels-Alder反应，3-甲基环丙烯将用于代谢性“光点击”反应。体外实验表征了它们的反应性和动力学。代谢标记将在HeLa细胞中通过RNA水解进行分析。结合FISH探针对选定的mRNA转录物和miRNA序列进行荧光特性评估。