Efficient Selection of DNA Aptamers with High Affinity and Specificity to GPCRs via Functional Extracellular Vesicles

Abstract Nucleic acid aptamers are promising new-generation experimental and therapeutic drugs for uncharacterized biomolecules and intractable diseases. However, efficient aptamer selection remains challenging. We present a novel functional selection method termed extracellular vesicle (EV)-SELEX that efficiently selects DNA aptamers for G protein-coupled proteins (GPCRs), a major class of drug targets via ligand-dependent GPCR endocytosis and subsequent release of GPCR-containing EVs. Using this method, we obtained Dapt-μR, a DNA aptamer that had a high affinity to μ-opioid receptor (MOR) (Kd ≈ 28 nM) but no other types of opioid receptors. In unmodified form, Dapt-μR acted as a morphine-like, naloxone-sensitive MOR agonist, inhibiting cAMP accumulation in cultured cells and reducing Ca²⁺ influx in primary striatal neurons. Furthermore, Dapt-μR selectively bound to the MOR-rich spinal cord dorsal horn and produced an analgesic effect following intrathecal administration in mice. These findings demonstrate that EV-SELEX is a powerful method for selecting DNA aptamers for target GPCRs.