Related Experiment Video
Updated: Jul 28, 2026
Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Published on: August 6, 2014
Simultaneous In Situ Detection of m6A-Modified and Unmodified RNAs Using DART-FISH
Charles J Sheehan1, Kate D Meyer2,3
1Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA.
Abstract:
N6-methyladenosine (m6A) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish m6A-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both m6A-modified and unmodified target transcripts. DART-FISH combines m6A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m6A stoichiometry and methylated mRNA localization in single cells.
Insights
Researchers developed DART-FISH, a new method to visualize N6-methyladenosine (m6A)-modified and unmodified mRNAs simultaneously within cells. This technique allows detailed study of m6A RNA localization and stoichiometry in single cells.
Area of Science:
- Molecular Biology
- Epigenetics
- RNA Biology
Background:
- N6-methyladenosine (m6A) is a prevalent mRNA modification crucial for RNA regulation and gene expression.
- Existing visualization methods cannot differentiate between m6A-modified and unmodified transcripts, hindering cellular localization studies.
- Understanding m6A distribution is vital for deciphering its functional roles in gene regulation.
Purpose of the Study:
- To introduce a novel technique, DART-FISH, for simultaneous visualization of m6A-modified and unmodified target mRNAs.
- To enable the investigation of m6A stoichiometry and the intracellular localization of methylated mRNAs.
- To overcome the limitations of traditional mRNA visualization methods.
Main Methods:
- DART-FISH combines m6A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization (FISH).
- This approach allows for the specific detection of both methylated and unmethylated mRNA copies.
- The technique is designed for single-cell analysis.
Main Results:
- Successful simultaneous detection of m6A-modified and unmodified target transcripts.
- Demonstration of DART-FISH's capability to analyze m6A stoichiometry.
- Visualization of methylated mRNA localization within single cells.
Conclusions:
- DART-FISH provides a powerful tool for studying m6A RNA biology at the single-cell level.
- The technique enhances our understanding of m6A modification's role in RNA function and gene expression.
- DART-FISH opens new avenues for investigating the spatial distribution and quantitative aspects of mRNA methylation.
