Structural mechanisms for binding and activation of a contact-quenched fluorophore by RhoBAST

Zhang, Yufan; Xu, Zhonghe; Xiao, Yu; Jiang, Haodong; Zuo, Xiaobing; Li, Xing; Fang, Xianyang

doi:10.1038/s41467-024-48478-9

Title: Structural mechanisms for binding and activation of a contact-quenched fluorophore by RhoBAST

Journal Article · 17 May 2024 · Nature Communications

DOI: https://doi.org/10.1038/s41467-024-48478-9 · OSTI ID:2470020

Zhang, Yufan ^[1];

^[2]; Xiao, Yu ^[2]; Jiang, Haodong ^[1]; Zuo, Xiaobing ^[3];

^[1];

^[4]

Chinese Academy of Sciences (CAS), Beijing (China)
Tsinghua Univ., Beijing (China)
Argonne National Laboratory (ANL), Argonne, IL (United States)
Chinese Academy of Sciences (CAS), Beijing (China); Tsinghua Univ., Beijing (China)

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.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: National Key Research and Development Program of China; National Natural Science Foundation of China (NSFC); USDOE

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2470020

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 15; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Title: Structural mechanisms for binding and activation of a contact-quenched fluorophore by RhoBAST

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