Functional requirements for specific ligand recognition by a biotin-binding RNA pseudoknot

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Aptamers, selected RNA or DNA molecules, serve as models for studying RNA structure-function relationships.
• Understanding ligand-specific aptamer structures is crucial for developing molecular tools.

Purpose of the Study:

• To define the structural requirements for high-affinity biotin binding by RNA aptamers.
• To investigate the conserved structural motif responsible for biotin recognition.

Main Methods:

• In vitro selection (SELEX) to isolate biotin-binding aptamers.
• Chemical probing to assess RNA structure and ligand-induced changes.
• Competition assays to determine binding epitope.
• Three-dimensional modeling to visualize ligand-aptamer interactions.

Main Results:

• A conserved pseudoknot motif, with an adenosine-rich loop, was identified in all selected biotin aptamers.
• Extensive base pairing (14 nucleotides) is critical for binding, with minimal contribution from unpaired nucleotides.
• Ligand binding induced minimal structural changes in the aptamer.
• Competition experiments indicated recognition of the entire biotin molecule.

Conclusions:

• The identified pseudoknot represents a near-global optimum for biotin recognition.
• Aptamer binding relies heavily on base-paired nucleotides, contrasting with other known aptamers.
• The structure facilitates comprehensive interactions with the biotin ligand.