Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell’s phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low- and high-density sEV subsets from a human mast cell line (HMC-1) and an erythroleukemic cell line (TF-1) were separated using high-resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV-associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high-density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole-genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology.

细胞外囊泡具有在细胞之间转移脂质、蛋白质和核酸的能力，从而影响受体细胞的表型。虽然 RNA 在 EV 中的作用已被广泛研究，但 DNA 的功能仍然难以捉摸。在这里，我们根据 DNA 含量和拓扑结构区分了小细胞外囊泡 (sEV) 的新型异质亚群。使用高分辨率碘克沙醇密度梯度分离来自人类肥大细胞系 (HMC-1) 和红白血病细胞系 (TF-1) 的低密度和高密度 sEV 子集，以区分 sEV 的 DNA 货物的性质。sEV 相关 DNA 和 RNA 分子的配对比较表明，RNA 比 DNA 更丰富，并且大多数 DNA 存在于高密度部分中，这表明 sEV 亚群具有不同的 DNA 含量。DNA 主要位于 sEV 的外部或表面，只有一小部分受到保护，免受酶促降解。当对 sEV 进行批量分析时，全基因组测序鉴定出跨越所有染色体的 DNA 片段和线粒体 DNA。我们的工作有助于理解 DNA 如何与 sEV 相关联，从而为根据 DNA 货物和拓扑区分 EV 亚型提供方向。