Homogeneous DNA sensing using enzyme-inhibiting DNA aptamers

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Enzyme-inhibiting aptamers offer precise control over enzymatic activity.
• Aptameric enzyme subunits (AES) are emerging tools for molecular detection.
• Thrombin-inhibiting aptamers with G-quartet structures are well-established for enzyme inhibition.

Purpose of the Study:

• To develop a novel aptameric enzyme subunit (AES) for sensitive and homogeneous detection of target DNA.
• To engineer an AES capable of allosterically modulating enzymatic activity based on target DNA binding.
• To establish a new DNA detection strategy with a low detection limit and without the need for separation.

Main Methods:

• Design and synthesis of a modified thrombin-inhibiting aptamer incorporating a target DNA-binding stem-loop structure.
• Integration of the stem-loop into the G-quartet core of the aptamer to create the AES.
• Assay development for homogeneous detection of target DNA by monitoring changes in thrombin activity.
• Determination of the lower detection limit for target DNA using the developed AES.

Main Results:

• A novel AES was successfully developed by inserting a target DNA-complementary strand into a thrombin-inhibiting aptamer.
• Target DNA binding induced hybridization and disrupted the aptamer's G-quartet structure, allosterically modulating thrombin activity.
• The AES enabled homogeneous detection of target DNA by measuring changes in thrombin activity.
• A low detection limit of 20 nM for target DNA was achieved.

Conclusions:

• The developed aptameric enzyme subunit (AES) provides a sensitive and homogeneous method for target DNA detection.
• This approach leverages allosteric control of enzymatic activity via aptamer structure modulation.
• The novel AES system eliminates the need for separation steps, simplifying the detection process.