Structural basis of a small monomeric Clivia fluorogenic RNA with a large Stokes shift.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RNA-based fluorogenic modules are crucial for tracking RNA molecules in real-time.
• The red-emitting fluorophore NBSI (5-((Z)-4-((2-hydroxyethyl)(methyl)amino)benzylidene)-3-methyl-2-((E)-styryl)-3,5-dihydro-4H-imidazol-4-one) and its aptamer Clivia offer a large Stokes shift.
• Understanding the Clivia-NBSI complex structure is key to optimizing its use in biological imaging.

Purpose of the Study:

• To determine the tertiary structure of the Clivia-NBSI RNA-fluorophore complex.
• To analyze the molecular basis of NBSI recognition by the Clivia aptamer.
• To develop enhanced RNA imaging tools for live-cell applications.

Main Methods:

• Tertiary structure determination of the Clivia-NBSI complex.
• Structure-based analysis of fluorophore recognition patterns.
• Fluorescence assays and structure-based substitution assays.

Main Results:

• The Clivia-NBSI complex adopts a compact coaxial architecture with NBSI at the core junction.
• Orthogonal use of Clivia-NBSI with other aptamers is enabled, allowing dual-emission imaging.
• A multivalent Clivia aptamer with enhanced sensitivity was developed for improved in vitro and in vivo fluorophore recognition.

Conclusions:

• The determined structure provides molecular insights into the Clivia-NBSI interaction.
• The developed aptamer designs enable advanced RNA imaging techniques in living cells.
• This work paves the way for efficient applications in biomedical research and diagnostics.