Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging

Daniel Englert, Eva-Maria Burger, Franziska Grün, Mrigank S. Verma, Jens Lackner, Marko Lampe, Bastian Bühler, Janin Schokolowski, G. Ulrich Nienhaus (), Andres Jäschke () and Murat Sunbul ()
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Daniel Englert: Heidelberg University
Eva-Maria Burger: Heidelberg University
Franziska Grün: Heidelberg University
Mrigank S. Verma: Karlsruhe Institute of Technology (KIT)
Jens Lackner: Karlsruhe Institute of Technology (KIT)
Marko Lampe: European Molecular Biology Laboratory
Bastian Bühler: Heidelberg University
Janin Schokolowski: Heidelberg University
G. Ulrich Nienhaus: Karlsruhe Institute of Technology (KIT)
Andres Jäschke: Heidelberg University
Murat Sunbul: Heidelberg University

Nature Communications, 2023, vol. 14, issue 1, 1-13

Abstract: Abstract Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer. High brightness and fluorogenicity is achieved by shifting the equilibrium between spirolactam and quinoid. With its high affinity and fast ligand exchange, RhoBAST:SpyRho is a superb system for both super-resolution SMLM and STED imaging. Its excellent performance in SMLM and the first reported super-resolved STED images of specifically labeled RNA in live mammalian cells represent significant advances over other FLAPs. The versatility of RhoBAST:SpyRho is further demonstrated by imaging endogenous chromosomal loci and proteins.

Date: 2023
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DOI: 10.1038/s41467-023-39611-1

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