Secondary structures formed by single-stranded DNA aptamers can allow for the binding of small-molecule ligands. Some of these secondary structures are highly stable in solution and are great candidates for use in the development of molecular tools for biomarker detection, environmental monitoring, and others. In this paper, we explored adenosine triphosphate (ATP)-binding aptamers for the simultaneous detection of two small-molecule ligands: adenosine triphosphate (ATP) and thioflavin T (ThT). The aptamer can form a G-quadruplex (G4) structure with two G-quartets, and our results show that each of these quartets is equally involved in binding. Using fluorescently labeled and label-free methods, we further explored the role of the G4 motif in modulating the ligand binding property of the aptamer by making two extended variants that can form three or four G-quartet G4 structures. Through equilibrium binding and electrospray ionization mass spectrometry (ESI-MS) analysis, we observed a stronger affinity of aptamers to ATP by the variant G4 constructs relative to the native aptamer (Kd range of 0.040-0.042 μM for variants as compared to 0.15 μM for the native ATP aptamer). Additionally, we observed a dual binding of ThT and ATP to the G4 constructs in the label-free and ESI-MS analyses. These findings together suggest that the G4 motif in the ATP aptamer is a critical structural element that is required for optimum ATP binding and can be modulated for the binding of multiple ligands. These findings are instrumental for designing smart molecular tools for a wide range of applications, including biomarker monitoring and ligand binding studies.
© 2024 The Authors. Published by American Chemical Society.