Structure Switchable Single Fluorophore Biosensor to Measure Dissociation Constant in PAT Aptamer Tailoring

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Aptamers offer high specificity and cost-effectiveness for biosensing but current methods are complex and expensive.
• Existing biosensing platforms often require specialized equipment and labor-intensive modifications.

Purpose of the Study:

• To develop a cost-effective, real-time quantitative PCR (qPCR)-based platform for aptamer affinity (K_d) determination.
• To engineer and validate a patulin aptamer for enhanced binding affinity.

Main Methods:

• Integration of agarose-immobilized targets with fluorescent light-up extension primers.
• Real-time quantitative PCR (qPCR) for K_d determination.
• Aptamer tailoring by removing primer regions and terminal bases, followed by molecular docking analysis.

Main Results:

• A modified aptamer (M1) showed a 2.5-fold increase in affinity (K_d = 33.41nM) compared to the original 78-mer aptamer.
• Method validation confirmed the platform's reliability and the aptamer engineering approach's efficacy.
• Molecular docking identified central stem GC base pairs as key interaction sites.

Conclusions:

• A cost-effective, standard qPCR-based platform for aptamer affinity quantification was established.
• This platform improves the quantitative assessment of aptamers derived from Systematic Evolution of Ligands by Exponential Enrichment (SELEX).
• The study provides a robust framework for advancing aptamer applications in biosensing.