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Updated: Jul 24, 2025

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
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Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging.

Daniel Englert1, Eva-Maria Burger1, Franziska Grün1

  • 1Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.

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|June 30, 2023
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Summary
This summary is machine-generated.

Researchers developed RhoBAST:SpyRho, a novel fluorescent light-up aptamer (FLAP) system for high-resolution live-cell RNA imaging. This system overcomes previous limitations, enabling clear visualization of RNA and other cellular components.

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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.