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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Published on: August 6, 2014

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

Moon Jung Kim¹, Yida Li¹, Jason A Junge^2,3

¹Department of Chemistry & Loker Hydrocarbon Research Institute, University of Southern California, Los Angeles, California 90089, United States.

ACS Chemical Biology

|May 18, 2023

Summary

New indolizine-based styrene dyes offer superior fluorescence for live cell RNA imaging. These probes provide intense staining, resolve subnucleolar structures, and visualize RNA coacervates, outperforming existing RNA dyes.

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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.