| dc.description.abstract | Aptamers are rare functional nucleic acids with binding affinity and specificity to target ligands. In past years, several high-resolution structures of aptamer complexes have shed light on the binding mode and recognition principles of aptamer complexes interactions. However, aptamer complex binding behavior and mechanism are not clearly understood especially with the absence of structural information. In this study, it was demonstrated that isothermal titration calorimetry (ITC), circular dichroism (CD) and molecular dynamics (MD) simulations were useful tools for studying the fundamental binding mechanism between DNA aptamers and small molecules. To gain further insight into this behavior, thermodynamic and conformational measurements under different parameters such as salt concentration, temperature, pH value were carried out.
In this study, we choose three DNA molecules to examine the binding behaviors between these DNA molecules and small ligands. The first system is to illustrate the binding mechanism of daunomycin binding with a simple dsDNA. The results suggest that the binding free energy more favorable with temperature increased; this is contributed by the binding entropy. Furthermore, the amine group on daunomycin contributes electrostatic interaction that induces the binding process. In addition, enthalpy–entropy compensation is also exhibited in the daunomycin–DNA binding mechanism.
Secondly, we examined the binding mechanism between L-Tyrosinamide (L-TyrNH2) and its aptamer. The thermodynamic signature along with the coupled CD spectral change suggests that this binding behavior is an enthalpy driven process. The results showed that the interaction is an induced fit binding. The amide group and phenolic hydroxyl group of the L-TyrNH2 play a vital role in this binding mechanism. In addition, it should be noted that Mg2+ not only improves binding affinity but also helps change the structure of DNA aptamer.
The last one is a comparative study of the DNA aptamer binding with L-argininamide (L-Arm) and its enantiomer (D-Arm). The thermodynamics study reveals that both L-Arm and D-Arm binding with the aptamer are an enthalpy driven and entropy cost process, and L-Arm binding with the aptamer involved induced-fit binding mechanism. The protonated amino group of both L-Arm and D-Arm participates in electrostatic interaction and this interaction is stronger for D-Arm than L-Arm binding with the aptamer. From the opposite behavior of the heat capacity change of the two enantiomers, we could suggest that L-Arm and D-Arm bind in different binding site of aptamer and resulted in different conformations of the binding complexes.
From previous studies, we found that induced-fit binding mechanism usually involved in the binding processes between aptamer and ligand. We used explicit solvent MD simulations to examine the critical bases involved in aptamer-L-Arm binding and the induced-fit binding process in atomic resolution. The simulation results revealed that three pairs of bases (C9-C16, G10-C16, and A12-C17) play important roles in aptamer-L-Arm binding and that aptamer-L-Arm binding adopts a geometry optimized through a general induced-fit process. The mechanism has the following characteristic stages: adsorption stage, binding stage and complex stabilization stage.
In addition, simulation results showed that the L-Arm binding location of the aptamer is different with D-Arm. From electrostatic interaction energy profile and hydrogen bonding analysis, the binding mechanisms of D-Arm and L-Arm are also different. These results are in agreement with the experiment inferences. This study used an easy, convenient method of performing a systemic study in recognition systems. This study also provides the information of aptamer-ligand binding mechanism, and shows the detail information of the binding pathway. It provides additional information about microscopic mechanisms useful for molecular design, molecular recognition, and the structural investigation from NMR and X-ray crystallography. This information can offer a guideline for molecular engineering in aptamer recognition design.
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