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

Area of Science:

• Molecular Biology
• Biophysics
• Microscopy

Background:

• Live-cell RNA imaging requires high spatial and temporal resolution, a persistent challenge in cell biology.
• Existing fluorescent probes often suffer from poor cell permeability, low brightness, and insufficient signal-to-background ratios.

Purpose of the Study:

• To develop an advanced fluorescent light-up aptamer (FLAP) system for superior live-cell RNA visualization.
• To overcome limitations of current probes and enable advanced fluorescence microscopy techniques.

Main Methods:

• Development of a novel probe, SpyRho (Spirocyclic Rhodamine), designed for high affinity binding to the RhoBAST aptamer.
• Utilizing equilibrium shift between spirolactam and quinoid forms to enhance brightness and fluorogenicity.
• Application in super-resolution microscopy techniques, including super-resolution single-molecule localization microscopy (SMLM) and stimulated emission depletion (STED) microscopy.

Main Results:

• RhoBAST:SpyRho demonstrated high brightness, fluorogenicity, and signal-to-background ratio.
• Achieved the first super-resolved STED images of specifically labeled RNA in live mammalian cells.
• Successfully visualized endogenous chromosomal loci and proteins, showcasing system versatility.

Conclusions:

• RhoBAST:SpyRho represents a significant advancement in live-cell RNA imaging, surpassing existing FLAP systems.
• The system's performance in SMLM and STED imaging opens new avenues for studying RNA dynamics and localization.
• Its versatility extends to imaging other cellular targets, highlighting its broad applicability in cell biology research.