Selective DNA encapsulation in extracellular vesicles of Saccharomyces cerevisiae

Aim: Extracellular vesicles (EVs) are emerging mediators of intercellular communication capable of transporting nucleic acids, including plasmid and genomic DNA. This study aimed to investigate the presence, enrichment, and protection of distinct DNA classes in EV-enriched fractions from Saccharomyces cerevisiae (S. cerevisiae). Methods: Two plasmids were analyzed: the high-copy YEp352 (2µ) and the centromeric pRS316. EV-enriched fractions were isolated from yeast culture supernatants, and the presence of plasmid, ribosomal [18S ribosomal DNA (RDN18)], and mitochondrial [cytochrome c oxidase subunit I (COX1)] DNA was assessed. DNase digestion assays were performed to evaluate DNA protection, and transformation assays were conducted using S. cerevisiae and Escherichia coli. Enrichment of endogenous vs. exogenously added plasmid DNA was also compared. Results: Both plasmids were strongly enriched in EV fractions relative to culture supernatants. However, only pRS316 exhibited partial resistance to DNase digestion, suggesting encapsulation, while YEp352 remained fully susceptible. Despite this protection, pRS316-associated EVs did not mediate transformation of yeast or bacterial recipients, indicating compromised integrity or inefficient delivery. Endogenously produced plasmid DNA showed significantly higher enrichment than exogenously added DNA. Ribosomal and mitochondrial DNA were also detectable in EVs but were highly sensitive to DNase degradation, indicating minimal protection. Conclusion: These findings demonstrate that selective DNA incorporation into EVs depends on both DNA type and intrinsic plasmid features. The results suggest that plasmid properties influence EV-mediated transport and protection, highlighting the selective and cargo-specific nature of DNA packaging in yeast EVs.