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

Biniuri, Yonatan; Albada, Bauke; Willner, Itamar


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 Kd = 31 ± 3 μM, whereas most of the aptamer mutants show lower affinity (higher Kd 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 Kd values and the MST-derived Kd 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 (Kd = 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.