Key Laboratory of RNA Science and Engineering, Institute of Biophysics Chinese Academy of Sciences, Beijing, China.
Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China.
Nat Commun. 2024 May 17;15(1):4206. doi: 10.1038/s41467-024-48478-9.
The fluorescent light-up aptamer RhoBAST, which binds and activates the fluorophore-quencher conjugate tetramethylrhodamine-dinitroaniline with high affinity, super high brightness, remarkable photostability, and fast exchange kinetics, exhibits excellent performance in super-resolution RNA imaging. Here we determine the co-crystal structure of RhoBAST in complex with tetramethylrhodamine-dinitroaniline to elucidate the molecular basis for ligand binding and fluorescence activation. The structure exhibits an asymmetric "A"-like architecture for RhoBAST with a semi-open binding pocket harboring the xanthene of tetramethylrhodamine at the tip, while the dinitroaniline quencher stacks over the phenyl of tetramethylrhodamine instead of being fully released. Molecular dynamics simulations show highly heterogeneous conformational ensembles with the contact-but-unstacked fluorophore-quencher conformation for both free and bound tetramethylrhodamine-dinitroaniline being predominant. The simulations also show that, upon RNA binding, the fraction of xanthene-dinitroaniline stacked conformation significantly decreases in free tetramethylrhodamine-dinitroaniline. This highlights the importance of releasing dinitroaniline from xanthene tetramethylrhodamine to unquench the RhoBAST-tetramethylrhodamine-dinitroaniline complex. Using SAXS and ITC, we characterized the magnesium dependency of the folding and binding mode of RhoBAST in solution and indicated its strong structural robustness. The structures and binding modes of relevant fluorescent light-up aptamers are compared, providing mechanistic insights for rational design and optimization of this important fluorescent light-up aptamer-ligand system.
荧光点亮适体 RhoBAST 与荧光团猝灭体四甲基罗丹明-二硝基苯胺具有高亲和力、超高亮度、显著的光稳定性和快速交换动力学，在超分辨率 RNA 成像中表现出优异的性能。在这里，我们确定了 RhoBAST 与四甲基罗丹明-二硝基苯胺复合物的共晶结构，以阐明配体结合和荧光激活的分子基础。该结构呈现出 RhoBAST 的不对称“ A ”样结构，具有半开放的结合口袋，在尖端容纳四甲基罗丹明的香豆素，而二硝基苯胺猝灭剂堆积在四甲基罗丹明的苯基上，而不是完全释放。分子动力学模拟显示出高度异质的构象集合，对于游离和结合的四甲基罗丹明-二硝基苯胺，均以接触但未堆叠的荧光团-猝灭剂构象为主导。模拟还表明，在 RNA 结合后，游离的四甲基罗丹明-二硝基苯胺中香豆素-二硝基苯胺堆叠构象的分数显著降低。这突出表明，从香豆素四甲基罗丹明上释放二硝基苯胺对于未猝灭 RhoBAST-四甲基罗丹明-二硝基苯胺复合物非常重要。使用 SAXS 和 ITC，我们在溶液中表征了 RhoBAST 的折叠和结合模式的镁依赖性，并表明其具有很强的结构稳健性。比较了相关荧光点亮适体的结构和结合模式，为该重要的荧光点亮适体-配体系统的合理设计和优化提供了机制见解。
