Probing ATP/ATP-Aptamer or ATP-Aptamer Mutant Complexes by Microscale Thermophoresis and Molecular Dynamics Simulations: Discovery of an ATP-Aptamer Sequence of Superior Binding Properties

Microscale thermophoresis (MST) is used to follow the dissociation constants corresponding to ATTO 488-labeled adenosine triphosphate (ATP) and the ATP-aptamer or ATP-aptamer mutants that include two binding sites for the ATP ligand. A set of eight ATP-aptamer mutants, where the thymidine bases, within the reported ATP binding aptamer sites, are substituted with cytosine bases, are examined. The MST-derived dissociation constant of ATP to the reported aptamer is K_d = 31 ± 3 μM, whereas most of the aptamer mutants show lower affinity (higher K_d values) toward the ATP ligand. One aptamer mutant reveals, however, a higher affinity toward the ATP ligand, as compared to the reported ATP-aptamer. Molecular dynamics and docking simulations identify the structural features that control the affinities of binding of the ATP ligand to the two binding sites associated with the ATP-aptamer or the ATP-aptamer mutants. The simulated structures suggest that H-bonds between the ATP ligand and G₉ and G₁₁ bases, within one binding domain, and the π-π interactions between G₆ and the ATP purine moiety and the pyrimidine ring, in the second binding domain, control the affinity of binding interactions between the ATP ligand and the ATP-aptamer or ATP-aptamer mutant. Very good correlation between the computed K_d values and the MST-derived K_d values is found. The ATP-aptamer mutant (consisting of A₁→ G, T₄ → C, T₁₂ → C, A₂₄ → G, and T₂₇ → C mutations) reveals superior binding affinities toward the ATP ligands (K_d = 15 ± 1 μM) as compared to the binding affinity of ATP to the reported aptamer. These features of the mutant are supported by molecular dynamics simulations.