Nucleic acid aptamers are single-stranded RNA or DNA molecules that bind to their targets with high specificity and affinity.Although their biomedical applications have been booming,it is still debatable whether an aptamer recognizes its target through "induced fit" or "conformational selection",a central question in molecular recognition.To address this question,an ATP-binding DNA aptamer was selected as a model system and the conformational properties of this aptamer with and without the presence of ATP were investigated by single-pair Forster resonance energy transfer (spFRET) spectroscopy.The single-molecule results indicate that the aptamer can fold into a double-stranded-like structure,similar to the ligand-bound conformation,even without the presence of ATP The folded structure is thermally stable at high salt concentrations and becomes rather dynamic at low salt concentrations.Although in the latter condition,the aptamer prefers unfolded structures,it can occasionally migrate to the folded conformation for a short time before being unfolded again.The binding of ATP to the aptamer stabilizes the folded structure,which populates the ligand-bound state of the aptamer,thus shifting the conformational equilibrium.Collectively,our data supports that the ATP-binding DNA aptamer recognizes ATP ligand through "conformational selection".