Immobilization-free SELEX for aptamer discovery targeting colorectal cancer-derived small extracellular vesicles.

BACKGROUND: Despite the increasing prominence of small extracellular vesicles (sEVs) and liquid biopsies for early cancer diagnosis, the development of high-performance molecular probes specifically targeting sEVs remains limited. In this study, we present a novel enzymatic digestion sEV-systematic evolution of ligands by exponential enrichment (EDGE-SELEX) strategy that eliminates the need for sEV immobilization, thereby preserving the native structural and biochemical characteristics of sEVs and better mimicking their clinical environment. RESULTS: Using the EDGE-SELEX approach combined with a post-selection optimization process, we successfully identified two novel aptamers, H7F-3 and H15F, exhibiting high affinity for colorectal cancer (CRC)-derived sEVs, with dissociation constants of 8.149 and 3.347 nM, respectively. Structural analysis suggested that the G6 motif plays an important role in aptamer-sEV binding. This motif also demonstrated potential for incorporation into split and blocking aptamer designs. Furthermore, we developed an aptamer-based loop-mediated isothermal amplification for sEV detection (ABLE) system, which achieved detection limit of 20 particles/µL for CRC-derived sEVs. CONCLUSIONS: Our findings demonstrate the applicability of SELEX technology to native sEVs and highlight the diagnostic potential of the identified aptamers for sEV-based cancer detection. The EDGE-SELEX method and the G6 motif may serve as valuable tools for future clinical applications in non-invasive cancer diagnostics and aptamer engineering, although further validation with clinical samples is warranted.