G-Quadruplex Structure in the ATP-Binding DNA Aptamer Strongly Modulates Ligand Binding Activity

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• DNA aptamers form secondary structures for small-molecule ligand binding.
• These structures are stable and useful for developing molecular tools in diagnostics and monitoring.
• Adenosine triphosphate (ATP) aptamers are explored for their binding capabilities.

Purpose of the Study:

• To investigate adenosine triphosphate (ATP)-binding aptamers for simultaneous detection of ATP and thioflavin T (ThT).
• To explore the role of the G-quadruplex (G4) motif in modulating aptamer ligand binding properties.
• To engineer aptamer variants with enhanced binding affinity and dual-ligand recognition.

Main Methods:

• Utilized fluorescently labeled and label-free detection methods.
• Constructed extended aptamer variants with increased G-quadruplex units (three or four G-quartets).
• Performed equilibrium binding assays and electrospray ionization mass spectrometry (ESI-MS) analysis.

Main Results:

• The native aptamer forms a G-quadruplex (G4) structure with two G-quartets, both involved in binding.
• Extended G4 aptamer variants exhibited significantly stronger affinity for ATP (Kd range 0.040-0.042 μM) compared to the native aptamer (0.15 μM).
• Label-free and ESI-MS analyses confirmed dual binding of both ThT and ATP to the engineered G4 constructs.

Conclusions:

• The G-quadruplex (G4) motif is crucial for optimal ATP binding in these aptamers.
• Modulating the G4 structure enhances ATP binding affinity and enables simultaneous binding of multiple ligands.
• These findings facilitate the design of advanced molecular tools for biomarker monitoring and ligand binding studies.