Structural basis for ring-opening fluorescence by the RhoBAST RNA aptamer.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Fluorogenic aptamers enable live-cell RNA imaging, advancing understanding of RNA metabolism.
• Recent advances utilize spirocyclic rhodamine dyes for robust performance in mammalian cells.
• These dyes switch from a non-fluorescent to a fluorescent state upon binding aptamers.

Purpose of the Study:

• To elucidate the structural basis of RhoBAST aptamer-mediated fluorogenicity with SpyRho555 dye.
• To understand the molecular interactions driving dye binding and fluorescence activation.
• To inform the rational design of next-generation fluorogenic aptamers.

Main Methods:

• X-ray crystallography was used to determine the structures of RhoBAST bound to dye analogues.
• Mutagenesis studies were performed to validate the role of specific residues in dye binding and activation.
• Biophysical techniques likely employed to characterize binding and fluorescence properties (implied).

Main Results:

• Crystal structures revealed the RhoBAST aptamer's four-way junction organizing a dye-binding pocket.
• The dye's core is stabilized by pi-stacking interactions with adenine and guanine bases.
• An unpaired guanine residue directly interacts with the dye, promoting its fluorescent, open conformation.

Conclusions:

• The RhoBAST aptamer structure explains SpyRho555 dye activation through specific base-dye interactions.
• A key guanine interaction stabilizes the dye's open, fluorescent state.
• These findings support structure-guided design for developing improved fluorogenic aptamers for RNA imaging.