Efficient and specific selection of high-affinity DNA aptamers targeting μ-opioid receptor via functional extracellular vesicles

Nucleic acid aptamers are promising next-generation experimental and therapeutic drugs for uncharacterized biomolecules and intractable diseases. However, efficient aptamer selection remains challenging. We have developed a functional selection method termed extracellular vesicle (EV)-SELEX that efficiently selects DNA aptamers for G protein-coupled receptors (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. In living cells, ligand-bound GPCRs are internalized, packaged into the extracellular vesicles, and released. By utilizing this pathway as a screening platform, one can efficiently generate a high-performance DNA aptamer drug for a specific GPCR.