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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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Programmable, Structure-Switching RhoBAST for Hybridization-Mediated mRNA Imaging in Living Cells.
Bastian Bühler1, Janin Schokolowski1, Andres Jäschke1
1Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, 69120 Heidelberg, Germany.
ACS Chemical Biology
|August 2, 2023
Summary
Researchers developed programmable RhoBAST aptamers to visualize untagged messenger RNAs (mRNAs) in living cells. These probes light up only when bound to specific target RNAs, enabling precise RNA imaging.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Visualizing endogenous RNAs in living cells is essential for understanding their functions.
- Fluorescence light-up aptamers (FLAPs) offer a promising approach for RNA imaging.
- RhoBAST is a highly photostable and bright FLAP developed for messenger RNA (mRNA) imaging.
Purpose of the Study:
- To engineer programmable RhoBAST sequences for specific, in situ detection of untagged target RNAs.
- To create a modular system where target binding induces RhoBAST conformational changes for fluorescence activation.
- To demonstrate the utility of programmable RhoBAST for live bacterial mRNA visualization.
Main Methods:
- Design of RhoBAST sequences with flanking hybridization arms containing a modular transducer.
- Characterization of structural switching and fluorescence light-up properties in vitro.
- Application of programmable RhoBAST for live imaging of untagged mRNAs in bacteria.
Main Results:
- Programmable RhoBAST sequences were successfully designed to bind specific target RNAs in trans.
- A modular transducer sequence controlled RhoBAST structure, enabling target-dependent fluorescence.
- The system demonstrated specific fluorescence light-up upon binding to target mRNA after interaction with TMR-DN.
- Successful visualization of untagged mRNAs in live bacterial cells was achieved.
Conclusions:
- Programmable RhoBAST aptamers provide a novel and specific method for imaging endogenous, untagged RNAs.
- This modular system offers versatility for targeting different RNA molecules.
- The developed probes are suitable for visualizing mRNA dynamics in live bacterial systems.
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