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Updated: Jun 2, 2025

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
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Structural basis for ring-opening fluorescence by the RhoBAST RNA aptamer.

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    New aptamers enable live-cell RNA imaging by unlocking the fluorescence of spirocyclic rhodamine dyes. Structural studies reveal how the RhoBAST aptamer binds dyes, stabilizing their fluorescent state for cellular RNA visualization.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Structural Biology

    Background:

    • Fluorogenic aptamers are crucial for live-cell RNA imaging, revealing insights into RNA metabolism and dynamics.
    • Recent advancements utilize spirocyclic rhodamine dyes for enhanced performance in mammalian cells.
    • These dyes switch between a non-fluorescent, cell-permeable state and a fluorescent state.

    Purpose of the Study:

    • To elucidate the structural basis for the fluorogenicity of the RhoBAST aptamer-SpyRho555 dye complex.
    • To understand the molecular interactions governing dye binding and fluorescence activation.

    Main Methods:

    • X-ray crystallography was used to determine the structures of the RhoBAST aptamer bound to dye analogs.
    • Mutagenesis studies were performed to validate the role of specific nucleotide-aptamer interactions.

    Main Results:

    • Crystal structures revealed the RhoBAST aptamer forms a four-way junction creating a dye-binding pocket.
    • The dye's core is stabilized by pi-stacking interactions with adenine bases.
    • A key guanine base interacts with the dye, stabilizing its fluorescent, open conformation.

    Conclusions:

    • The RhoBAST aptamer's structure facilitates dye binding and fluorescence.
    • Specific base-pairing interactions, particularly with guanine, are critical for activating dye fluorescence.
    • This work provides a structural foundation for developing advanced RNA imaging tools.