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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Published on: August 6, 2014
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Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging.
Bastian Bühler1, Janin Schokolowski1, Anja Benderoth1
1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.
Nature Chemical Biology
|January 19, 2023
Summary
Researchers developed brighter fluorescent light-up aptamers (FLAPs) for RNA visualization. These enhanced FLAPs offer improved brightness, photostability, and lower background, enabling robust mRNA imaging and tracking in live cells.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Fluorescent light-up aptamers (FLAPs) are crucial for RNA visualization in live cells.
- Existing FLAPs suffer from low brightness, poor photostability, and high background fluorescence.
- Limitations hinder effective RNA imaging and tracking applications.
Purpose of the Study:
- To develop novel FLAPs with enhanced brightness and photostability.
- To improve aptamer-dye interactions and fluorogenicity for superior signal.
- To enable simultaneous imaging of multiple RNA species and single-molecule RNA dynamics.
Main Methods:
- Exploited the avidity concept to design dimeric aptamers (biRhoBAST, biSiRA) with bivalent ligands (TMR2, SiR2).
- Constructed RNA scaffolds to embed the dimeric aptamer units.
- Tested red (biRhoBAST-TMR2) and near-infrared (biSiRA-SiR2) fluorescent FLAPs for RNA imaging in live cells.
Main Results:
- Developed biRhoBAST-TMR2 and biSiRA-SiR2, exhibiting exceptional brightness and photostability.
- Achieved significantly higher signal-to-background ratios for mRNA imaging compared to existing FLAPs.
- Demonstrated orthogonal imaging of two RNA species and single-molecule mRNA tracking with minimal perturbation.
Conclusions:
- Avidity-enhanced FLAPs represent a significant advancement in RNA visualization technology.
- These new FLAPs overcome limitations of previous tools, offering robust and sensitive RNA imaging.
- The developed FLAPs hold great potential for studying RNA dynamics in live cells.
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