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

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 designed 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.

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small‐molecule Fluorophores

“…Höbartner lab reported Chili: DMHBI system [39] . Sunbul and Jäschke's labs reported RhoBAST: SpyRho [40] . Yang lab reported Pepper: HBC system [9] et al.…”

“…In addition, the imaging systems were constructed as various RNA‐based sensors for small‐molecule metabolite, protein, ions detection in living cells [42] . Moreover, the systems can be engineered for chromosomal imaging with CRISPR [9, 40, 43] …”

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small‐molecule Fluorophores

“…In addition, the imaging systems were constructed as various RNA‐based sensors for small‐molecule metabolite, protein, ions detection in living cells [42] . Moreover, the systems can be engineered for chromosomal imaging with CRISPR [9, 40, 43] …”

“…Höbartner lab reported Chili: DMHBI system [39] . Sunbul and Jäschke's labs reported RhoBAST: SpyRho [40] . Yang lab reported Pepper: HBC system [9] et al.…”

“…[39] Sunbul and Jäschke's labs reported RhoBAST: SpyRho. [40] Yang lab reported Pepper: HBC system [9] et al As Corn and Beetroot are dimers and unlikely designed for single mRNA imaging, other systems may have the potential for single mRNA imaging with advanced microscopes technique or the optimized conditions in the future.…”