On a nucleic acid-stained gel, clear DNA bands were visualized, and these were present not in samples pretreated with DNase-I but in samples treated with RNAse (Fig. 1A). The EVs fraction isolated from the density gradients had a charge of -70 mV, which was increased to -50 mV by DNase-I treatment (Fig. 1B). This result indicates that DNA is present on the outside of the EVs, and this DNA contributes to the negative charge of the vesicles. Further, DNase treatment increased the number of particles measured by nanoparticle tracking analysis, suggesting that the DNA to a certain degree contributes to aggregation of the isolated EVs (Fig. 1C). As a biological readout, isolated EVs associated with extracellular DNA were taken up by human mesenchymal stem cell in a time-dependent manner (Fig. 1D and E).
EVs carry multiple bioactive molecules, including proteins, various RNA species, and according to the present study, DNA. In our case, the DNA was observed in EV isolates from a human mast cell line. Specifically, our study argues that the DNase-sensitive nucleic acids are present on the outside of the EVs. Furthermore, the EV-associated DNA can be taken up by recipient cells, which could alter cellular responses.
It is known that EVs can mediate an array of biological messages to recipient cells- including surface-to-surface antigen presentation and receptors activation- and can deliver RNA (e.g. mRNA, miRNA) cargo to recipient cells. However, the presence and function of DNA as a cargo on the outside of EVs is less explored. EVs have previously been associated with cell-free DNA that carries retrotransposon elements and oncogenes, but overall EV-associated DNA has been extensively characterized. A recent report emphasizes the presence of dsDNA inside of the EVs, whereas we find that a majority of DNA from the human mast cell line is associated with outer perimeter of EVs, since it is sensitive to DNase treatment without lysing the EVs. Our study also indicates, for the first time, that DNA covering floated EVs can lead to an increase in the net negative charge of the vesicles. This was confirmed by reduction of net negative charge from EVs by DNase treatment. We were also able to monitor the increase in particle numbers after DNase treatment, indirectly suggesting that EV-DNA may lead to aggregation of EVs. These results are in line with previous observation made in various electron micrographs, showing clustering of EVs, which could have occurred because of DNA on the surface of these vesicles. The aggregation of EVs may be secondary to nonspecific aggregation of EVs during ultracentrifugation, but our study suggests that EV-related DNA can contribute to this observation. As we observed that the extracellular DNA floated in the density gradient, we argue strongly that it is associated with the floating vesicles with relatively low density. Also, it is has been shown that exogenous plasmids DNA if associated with EVs are taken up more efficiently in recipient cells than free DNA, again arguing that association to EVs could be involved in DNA uptake. Similarly, we also observed a time-dependent increase in cytoplasmic DNA foci in EV treated recipient human mesenchymal stem cells. Overall, this study highlights the need to define the EV-associated DNA in delivering biological function in cells that take up these EVs.