Selection of Aptamers Targeting Different Epitopes of Recombinant Thyroid-Stimulating Hormone Receptor Protein

An epitope is the portion of an antigen that binds to specific B cell receptors and recognition molecules like antibodies and aptamers. Identifying different epitopes aids in diagnostic biomarker discovery, vaccine development, and therapeutic target identification, thus advancing precision medicine. Aptamers, generated through SELEX technology, are effective target recognition elements. However, the selection of aptamers targeting native epitopes on clinically relevant targets remains a significant challenge primarily due to the scarcity of tag-free, natively folded protein preparations. Herein, we developed a novel SELEX method and identified aptamers targeting different epitopes of the recombinant thyroid-stimulating hormone receptor (TSHR) protein. By immobilizing target molecules with an epitope-I-specific aptamer, we generated aptamers recognizing other natural TSHR epitopes (epitope-II). Testing at the cellular and tissue levels confirmed the specificity and affinity of these aptamers for TSHR binding. Flow cytometry and immunofluorescence further validated their ability to capture TSHR protein epitopes, indicating potential as specific molecular probes. Immunohistochemistry on thyroid tumor samples demonstrated the diagnostic utility of multiple aptamers in identifying TSHR-positive pathology. This research advances aptamer-based diagnostics and therapeutics for thyroid cancer and offers a generalizable method for developing aptamers against multiple protein epitopes, accelerating biomarker and therapeutic target discovery.