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

## TL;DR

A new SELEX method was developed to create aptamers that bind to colorectal cancer-derived extracellular vesicles, offering potential for non-invasive cancer detection.

## Contribution

The novel EDGE-SELEX strategy eliminates sEV immobilization, preserving their native state for better aptamer discovery.

## Key findings

- Two novel aptamers with high affinity for CRC-derived sEVs were identified.
- The G6 motif was found to be important for aptamer-sEV binding and aptamer design.
- An ABLE system detected CRC-derived sEVs at a limit of 20 particles/µL.

## Abstract

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.

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.

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.

The online version contains supplementary material available at 10.1186/s12951-025-03813-0.