(1. College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410000, China; 

2. College of Biology, Hunan University, Changsha 410000, China)

Abstract: Extracellular vesicles (EVs) were isolated from the culture supernatant of lung adenocarcinoma A549 cells. The size of EVs was characterized as approximately 100 nm via DLS and TEM analysis, with the EV-specific marker TSG101 detected in the samples. The extracted EV DNA (evDNA) showed a predominantly～12 kb size distribution as assessed by agarose gel electrophoresis. Based on sequencing data, specific evDNA was selected for PCR detection. DNase I or PS nuclease treatment of the EVs confirmed that the evDNA existed on the outside of the vesicle in a linear manner, and poly-dA tails were added to evDNA by TdT enzyme treatment, allowing for their capture by oligo-dT beads and presenting further evidence of their linear surface distribution. Additionally, a TetO-DNA/TetR-GFP visualization system was established to confirm the presence of TetO-DNA in the EVs, which could be visualized by TetR-GFP proteins under fluorescent microscopy. Enrichment of EVs carrying the TetO-DNA was confirmed via flow cytometry and PCR experiments using TetR-GFP protein. These findings collectively suggest that evDNA is distributed linearly on the surface of EVs, providing a basis for further research into their biological functions.

Key words: lung adenocarcinoma cells; extracellular vesicles; evDNA; TetO-DNA; TetR-GFP

（Acta Laser Biology Sinica, 2024, 33(2): 151-159）