DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology

ABSTRACT

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.

KEYWORDS:

• Small extracellular vesicles
• sEVs
• exosomes
• extracellular DNA
• cell-free DNA
• DNA topology
• histones
• density gradient

Acknowledgments

The authors acknowledge support from the National Genomics Infrastructure in Stockholm funded by the Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, and the Swedish Research Council and acknowledge the SNIC/Uppsala Multidisciplinary Centre for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. We want to thank Gunnar Nilsson at the Karolinska Institute, Stockholm, Sweden, for the kind gift of the human mast cell line HMC-1. The authors would like to acknowledge data analysis support from the Bioinformatics Core Facility at the Sahlgrenska Academy, University of Gothenburg, and microscopy support from the Centre for Cellular Imaging at the University of Gothenburg and the National Microscopy Infrastructure, NMI (VR-RFI 2016-00968). The authors acknowledge Lukas Badertscher for his assistance with graphic design. We thank the Proteomics Core Facility at Sahlgrenska Academy, University of Gothenburg University, for performing the proteomic analysis.

Declaration of Interests

ELI is currently employed and holds equity in AstraZeneca R&D. SCJ is presently employed by Codiak BioSciences Inc., USA. JL holds equity in Codiak BioSciences, Inc. JL, ELI, CL, and SCJ are inventors of patents using EVs as either therapeutic or diagnostics tools.

Supplementary material

Supplemental data for this article can be accessed here

Additional information

Funding

This work was funded by grants from the Swedish Research Council, the Swedish Cancer Foundation, VBG Group Herman Krefting Foundation for Asthma and Allergy Research, and the Swedish Heart and Lung Foundation. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors also thank the Cost Action BM1202 MEHAD for the Short-Term Scientific Missions (STSMs) Grant (ME-HaD) for ELI to visit the Krefting Research Centre at the University of Gothenburg;Hjärt-Lungfonden;Cost Action BM1202 MEHAD for the Short Term Scientific Missions (STSMs) Grant (ME-HaD).