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

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.