Aptameric enzyme subunit for biosensing based on enzymatic activity measurement

The aptameric enzyme subunit (AES), which is a DNA aptamer composed of an enzyme-inhibiting aptamer and a target molecule-binding aptamer, has been developed for the biosensing of target molecules. We used a thrombin-inhibiting aptamer as the aptamer that inhibits enzymatic activity. The thrombin-inhibiting aptamer folds into the G-quartet structure, which plays an important role in its inhibitory activity. As a target molecule-binding aptamer, an adenosine-binding aptamer was inserted into the G-quartet structure of the thrombin-inhibiting aptamer to enable the change of the G-quartet structure upon the recognition of adenosine. In the present study, the change in the G-quartet structure led to a change in the thrombin inhibition activity, and adenosine was successfully detected by measuring the thrombin activity in a homogeneous solution without bound/free separation. We constructed two kinds of AESs; one of the structures is universal and can be used for designing any target molecule-binding aptamer. Since the enzyme activity is measured, AESs enable the simple and high-sensitivity detection of target molecules in a homogeneous assay.