Visualizing orthogonal RNAs simultaneously in live mammalian cells by fluorescence lifetime imaging microscopy (FLIM)

Visualization of RNAs in live cells is critical to understand biology of RNA dynamics and function in the complex cellular environment. Detection of RNAs with a fluorescent marker frequently involves genetically fusing an RNA aptamer tag to the RNA of interest, which binds to small molecules that ar...

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Bibliographic Details
Published in:Nature communications 2023-02, Vol.14 (1), p.867-867, Article 867
Main Authors: Sarfraz, Nadia, Moscoso, Emilia, Oertel, Therese, Lee, Harrison J., Ranjit, Suman, Braselmann, Esther
Format: Article
Language:English
Subjects:
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631/80/2373
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Animals
Aptamers
Bacteria - metabolism
Fluorescence
Fluorescent Dyes - metabolism
Fluorescent indicators
Humanities and Social Sciences
Mammalian cells
Mammals - metabolism
Microscopy
Microscopy, Fluorescence
multidisciplinary
Multiplexing
Nucleotide sequence
Ribonucleic acid
Riboswitch - genetics
RNA
RNA - genetics
RNA - metabolism
Science
Science (multidisciplinary)
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Visualization
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Summary:Visualization of RNAs in live cells is critical to understand biology of RNA dynamics and function in the complex cellular environment. Detection of RNAs with a fluorescent marker frequently involves genetically fusing an RNA aptamer tag to the RNA of interest, which binds to small molecules that are added to live cells and have fluorescent properties. Engineering efforts aim to improve performance and add versatile features. Current efforts focus on adding multiplexing capabilities to tag and visualize multiple RNAs simultaneously in the same cell. Here, we present the fluorescence lifetime-based platform Riboglow-FLIM. Our system requires a smaller tag and has superior cell contrast when compared with intensity-based detection. Because our RNA tags are derived from a large bacterial riboswitch sequence family, the riboswitch variants add versatility for using multiple tags simultaneously. Indeed, we demonstrate visualization of two RNAs simultaneously with orthogonal lifetime-based tags. No multi-color RNA fluorescent tags are currently available for use in live cells. Here, the authors show that fluorescence lifetime imaging microscopy is advantageous for multiplexed RNA visualization while achieving robust cellular contrast.
ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-023-36531-y