A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells.

Area of Science:

• Molecular Biology
• Biophysics
• Cell Biology

Background:

• Live-cell RNA imaging is crucial but limited by the lack of naturally fluorescent RNA molecules.
• Existing RNA aptamer-based methods suffer from insufficient brightness and photostability.
• Overcoming these limitations is key for advancing real-time RNA dynamics studies.

Purpose of the Study:

• To develop a novel, highly bright, and photostable fluorogenic system for live-cell RNA imaging.
• To enable sensitive detection of specific RNA species, including transcription products and mRNAs, in live mammalian cells.
• To establish a new design paradigm for next-generation ultrabright RNA probes.

Main Methods:

• Development of a cell-permeable fluorogenic dye dimer (Gemini-561) and a complementary dimerized aptamer (o-Coral).
• Utilizing the Gemini-561/o-Coral complex for direct fluorescence imaging of RNA polymerase III transcription products and messenger RNAs.
• Demonstrating imaging in live mammalian cells with single-copy aptamer labeling, avoiding tag multimerization.

Main Results:

• The Gemini-561/o-Coral complex exhibits significantly enhanced brightness and photostability compared to existing methods.
• Successful direct fluorescence imaging of RNA polymerase III transcription products and messenger RNAs in live mammalian cells.
• Achieved sensitive and fast detection of RNA with single-copy aptamer labeling.

Conclusions:

• The developed fluorogenic module provides a powerful tool for fast and sensitive intracellular RNA detection.
• The Gemini-561/o-Coral system overcomes previous limitations in RNA imaging, offering improved performance.
• This design concept paves the way for developing a new generation of ultrabright RNA probes for advanced biological research.