On May 17th, the research group led by Fang Xianyang from the Institute of Biophysics, Chinese Academy of Sciences, in collaboration with the research group led by Li Xing from the Institute of Zoology, published a research paper in Nature Communications. The study uncovers the mechanism of how the fluorescent RNA aptamer RhoBAST binds to and activates the fluorescent dye TMR-DN, shedding light on the rational design and optimization of this important FLAP system.
Precisely locating and tracking biomolecules such as proteins and RNA in living cells is crucial for understanding their biological functions and revealing their mechanisms. Currently, fluorescent labeling of RNA aptamers (FLAP) is considered a powerful tool for live-cell RNA fluorescence imaging. Among them, RhoBAST stands out as an excellent FLAP imaging tool, capable of binding to and activating a contact quenching-type rhodamine derivative TMR-DN, demonstrating outstanding performance in super-resolution RNA imaging. However, the structural mechanism underlying the binding and activation of the fluorescent dye is not yet clear.
Researchers first used X-ray crystallography to resolve the high-resolution structure of the RhoBAST-TMR-DN complex, revealing that RhoBAST folds into a four-branched structure resembling an asymmetric "A" shape. The study found that the structural rigidity of RhoBAST and the characteristics of its semi-open structural pocket may endow it with the dynamic capability of rapid ligand exchange, at least two orders of magnitude faster than other FLAP systems like the pepper system. This rapid exchange with fresh ligands in solution effectively avoids photobleaching issues, showcasing excellent optical properties for fluorescence super-resolution imaging.
To better understand the activation mechanism of RhoBAST on TMR-DN, researchers employed enhanced sampling molecular dynamics simulations to characterize the conformational space of TMR-DN in both free and bound states with RhoBAST. The results indicate that whether in the free or bound state, TMR-DN exhibits highly dynamic conformational features, with the contact-non-stacking conformation dominating among all contact types.
For more information on the research paper, visit: https://doi.org/10.1038/s41467-024-48478-9
