Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology, Goethe University, Max-von-Laue-Str. 7, 60438, Frankfurt/M., Germany.
National Centre for Biomolecular Research, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Angew Chem Int Ed Engl. 2021 Jan 11;60(2):865-872. doi: 10.1002/anie.202007184. Epub 2020 Nov 9.
We report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2'-deoxyguanosine to the aptamer domain of the bacterial 2'-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, we show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Our data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intracellular space. Here, we show that the in-cell NMR setup can be adjusted for characterization of much larger (≈70 nt) functional and chemically non-modified RNA.
我们在此报告了在真核细胞中，即非洲爪蟾卵母细胞和人宫颈癌细胞中，对细菌 2'-脱氧鸟苷感应核糖开关的适体结构域与天然配体 2'-脱氧鸟苷结合的细胞内 NMR 光谱观察。在这两种细胞类型中，核糖开关都足够稳定，可以检测到配体与核糖开关的结合。最重要的是，我们表明，通过体外对该原核核糖开关的特性研究建立的结合模式在真核细胞环境中得以维持。我们的数据还带来了重要的方法学见解：到目前为止，哺乳动物细胞内的 RNA 细胞内 NMR 研究仅限于对经过广泛保护基团修饰的短（<15nt）RNA 片段的研究，以限制其在细胞内空间中的降解。在这里，我们表明可以调整细胞内 NMR 装置以表征更大（≈70nt）的功能和化学非修饰 RNA。
