Evaluation of extracellular DNA distribution in bioliquids: exosome-associated vs. free circulating form

Exosomes contain a variety of biomolecules including DNA. Free circulating pool of extracellular DNA in bioliquids is another compartment that has a different origin and biochemistry. Knowledge of extracellular DNA distribution and localization in bioliquid is important for understanding both biological function of cfDNA and exosomes. Published data is still controversial and according to some publications up to 90% of cell-free blood DNA is associated with plasma exosomes. To quantify circulating DNA content in free vs. exosomal form in human plasma, urine, and saliva, we employed SubX technology, which allows affinity capture DNA via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. SubX simultaneously binds to both cfDNA and exosomes in bioliquids, thus allowing precipitation of the [SubX-DNA/SubX-Exosomes] complexes without ultracentrifugation. Intact exosomes can be reconstituted from the pellet in a low salt solution, while cfDNA remains insoluble as [SubX-DNA] precipitate. To evaluate solubility of [DNA-SubX] pellet in exosome reconstitution buffer sonicated DNA was spiked in PBS, then DNA was isolated and measured using Qubit^TM and qPCR. Exosomes step does not extract DNA form the [SubX-DNA] pellet and thus does not contaminate reconstituted exosomes with cfDNA. Over 90% of DNA in plasma and urine exist as a free circulating pool, while in saliva up to 30% is associated with exosomes. Thus, cfDNA distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfDNA-outer exosomal membrane aggregation. Since SubX tightly binds DNA and squeezes out proteins including histones from DNP complex, our approach allows for detaching cfDNA molecules non-specifically associated with exosome outer membrane in contrast to the PEG-based cfDNA and exosomes separation. Therefore, we suggest that highly-specific SubX technology allows for separation of cfDNA and exosomal DNA and thus precisely quantify extracellular DNA in different bioliquid compartments.