Bereiter Raphael, Flemmich Laurin, Nykiel Kamila, Heel Sarah, Geley Stephan, Hanisch Malou, Eichler Clemens, Breuker Kathrin, Lusser Alexandra, Micura Ronald
University of Innsbruck, Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), Innsbruck, Austria.
Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.
Nat Chem Biol. 2025 Jan 6. doi: 10.1038/s41589-024-01801-3.
Covalent labeling of RNA in living cells poses many challenges. Here we describe a structure-guided approach to engineer covalent RNA aptamer-ligand complexes. The key is to modify the cognate ligand with an electrophilic handle that allows it to react with a guanine at the RNA binding site. We illustrate this for the preQ-I riboswitch, in vitro and in vivo. Further, we demonstrate the versatility of the approach with a covalent fluorescent light-up aptamer. The coPepper system maintains strong fluorescence in live-cell imaging even after washing, can be used for super-resolution microscopy and, most notably, is uniquely suited for fluorescence recovery after photobleaching to monitor intracellular RNA dynamics. In addition, we have generated a Pepper ligand with a second handle for bioorthogonal chemistry to allow easily traceable pull-down of the covalently linked target RNA. Finally, we provide evidence for the suitability of this tethering strategy for drug targeting.
在活细胞中对RNA进行共价标记面临诸多挑战。在此，我们描述一种基于结构指导的方法来构建共价RNA适配体-配体复合物。关键在于用一个亲电基团修饰同源配体，使其能够与RNA结合位点的鸟嘌呤发生反应。我们在体外和体内针对preQ-I核糖开关对此进行了说明。此外，我们用一种共价荧光点亮适配体展示了该方法的多功能性。coPepper系统即使在洗涤后在活细胞成像中仍保持强烈荧光，可用于超分辨率显微镜，最值得注意的是，它特别适用于光漂白后的荧光恢复以监测细胞内RNA动态。此外，我们还生成了一种带有第二个用于生物正交化学基团的Pepper配体，以便轻松地对共价连接的靶RNA进行可追踪的下拉操作。最后，我们提供了该连接策略适用于药物靶向的证据。
