No Structure-Switching Required: A Generalizable Exonuclease-Mediated Aptamer-Based Assay for Small-Molecule...

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Small molecule binding to DNA can alter its susceptibility to exonuclease digestion.
• Aptamers, short DNA or RNA sequences, are used for molecular recognition and detection.

Purpose of the Study:

• To develop a sensitive, label-free method for small molecule detection using aptamers and exonucleases.
• To demonstrate the broad applicability and multiplexing capabilities of this novel assay.

Main Methods:

• Utilizing prefolded aptamers that resist exonuclease III digestion upon target binding.
• Employing a dual-exonuclease system (Exonuclease III and Exonuclease I) for selective product generation.
• Quantifying remaining double-stranded DNA products using fluorescent dyes like SYBR Gold.

Main Results:

• Target binding inhibits exonuclease III digestion, leaving a detectable double-stranded product.
• Nontarget-bound aptamers are degraded, while target-bound aptamers remain intact.
• Demonstrated sensitive detection of dehydroisoandrosterone-3-sulfate, cocaine, and ATP, including simultaneous multiplexed detection.

Conclusions:

• This dual-exonuclease aptamer assay provides a general, sensitive, and label-free platform for small molecule detection.
• The method is adaptable to various aptamers and targets, suitable for biological matrices.
• Potential for integration with diverse signal reporting strategies for broad analytical applications.