Mieczkowski Mateusz, Steinmetzger Christian, Bessi Irene, Lenz Ann-Kathrin, Schmiedel Alexander, Holzapfel Marco, Lambert Christoph, Pena Vladimir, Höbartner Claudia
Julius-Maximilians-Universität Würzburg, Institut für Organische Chemie, Am Hubland, 97074, Würzburg, Germany.
Georg August University School of Science, GGNB Doctoral Program Biomolecules, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
Nat Commun. 2021 Jun 10;12(1):3549. doi: 10.1038/s41467-021-23932-0.
Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO and DMHBI ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.
荧光RNA适配体是一类能特异性结合并激活条件荧光团的合成功能性RNA。Chili RNA适配体模仿了大斯托克斯位移荧光蛋白，对3,5-二甲氧基-4-羟基苯亚甲基咪唑啉酮（DMHBI）衍生物具有高亲和力，可发出绿色或红色荧光。在此，我们通过晶体学和时间分辨光谱学阐明了荧光激活的结构和机制基础。Chili RNA与带正电荷的DMHBO和DMHBI配体的两种共晶体结构揭示了一个G-四链体和一个通过π-π堆积固定配体的反式糖-糖边缘G:G碱基对。荧光团结合位点中的一个沃森-克里克G:C碱基对在鸟嘌呤的N7和配体的酚羟基之间建立了一个短氢键。发现从中性发色团到RNA的超快激发态质子转移（ESPT）的时间常数为130飞秒，揭示了大斯托克斯位移荧光RNA适配体的作用模式。
