Development and Methodological Refinement of SELEX protocol for RNA Aptamers targeting Melanoma-derived Extracellular Vesicles

Metastatic dissemination is the leading cause of cancer mortality, largely because secondary tumors are detected only after colonizing distant organs. Prior to dissemination, primary tumors prepare these sites by establishing a supportive microenvironment known as the pre-metastatic niche (PMN). Melanoma-derived extracellular vesicles (tEVs) drive PMN formation by traveling through the circulation to condition target tissues. Exploiting tEVs as an endogenous targeting system, using high-affinity RNA aptamers designed to bind tEVs and follow their natural homing axis into the PMN, presents a promising dual-action theranostic strategy. To generate these aptamers, this study aimed to establish an iterative SELEX (Systematic Evolution of Ligands by EXponential enrichment) workflow paired with high-sensitivity flow cytometry. Following vesicle characterization, selection rounds were conducted using clinical plasma samples, alternating negative selection against healthy donor EVs with positive selection against melanoma tEVs. Monitoring selection progress exposed critical technical bottlenecks: short, promoter-bearing PCR artifacts rapidly outcompeted the full-length library during amplification, producing false-positive transcript yields that masked target sequence loss. To resolve this, gentle MWCO partitioning and strict PCR cycle controls were evaluated to preserve EV membrane integrity and maintain library diversity, establishing an optimized framework for future EV-SELEX campaigns.