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 mu M, 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 G(9) and G(11) bases, within one binding domain, and the pi-pi interactions between G(6) 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 A(1)-> G, T-4 -> C, T-12 -> C, A(24) -> G, and T-27 -> C mutations) reveals superior binding affinities toward the ATP ligands (K-d = 15 +/- 1 mu M) as compared to the binding affinity of ATP to the reported aptamer. These features of the mutant are supported by molecular dynamics simulations.