Development of Highly Fluorogenic Styrene Probes for Visualizing RNA in Live Cells

Area of Science:

• Chemical Biology
• Molecular Imaging
• Biophysical Chemistry

Background:

• Styrene dyes are valuable fluorescent probes and sensors.
• Indole-containing styrene dyes selectively bind RNA but have limitations in fluorescence and background.
• Existing green-emitting indole dyes exhibit moderate fluorescence enhancement and quantum yields.

Purpose of the Study:

• To develop novel indole-based styrene dyes with improved imaging capabilities.
• To investigate positional and electronic effects of electron donors on dye properties.
• To create superior alternatives to current RNA-selective fluorescent dyes.

Main Methods:

• Synthesis of regioisomeric and isosteric analogues of indole-based styrene dyes.
• Evaluation of spectral properties (Stokes shift, molar extinction coefficient, wavelengths).
• Assessment of membrane permeability, fluorogenic response to RNA, FLIM compatibility, cytotoxicity, and photostability.

Main Results:

• Indolizine analogues showed enhanced spectral properties and high membrane permeability.
• These dyes exhibited strong fluorogenic responses upon RNA binding, low cytotoxicity, and excellent photostability.
• Indolizine dyes enabled rapid, sensitive nucleoli staining, resolved subnucleolar structures, and visualized RNA coacervates.

Conclusions:

• Indolizine-containing styrene probes offer the highest fluorescence enhancement among reported RNA-selective dyes.
• These novel dyes are effective for live-cell RNA visualization, resolving intricate cellular structures.
• They serve as excellent alternatives to SYTO RNASelect for both live-cell and in vitro RNA imaging.