Identification of eccDNA in Extracellular Vesicles Derived from Human Dermal Fibroblasts Through Nanopore Sequencing

Area of Science:

• Cell Biology
• Genomics
• Biochemistry

Background:

• Extrachromosomal circular DNAs (eccDNAs) are DNA molecules found outside chromosomes, implicated in cellular processes.
• Extracellular vesicles (EVs) are crucial for intercellular communication and contain various biomolecules, including nucleic acids.
• The presence and role of eccDNA within EVs remain largely unexplored.

Purpose of the Study:

• To investigate the presence of eccDNA within extracellular vesicles derived from human dermal fibroblasts.
• To characterize the eccDNA found in these EVs and their donor cells.
• To establish a framework for analyzing eccDNA in EVs for future research.

Main Methods:

• Isolation and sequencing of eccDNA from human dermal fibroblast-derived EVs and donor cells using Oxford Nanopore MinION.
• Bioinformatic analysis utilizing four distinct pipelines (ecc_Finder, cyrcular-calling, CReSIL, Flec) for eccDNA identification.
• Characterization of eccDNA size, chromosomal distribution, and gene fragment content.

Main Results:

• Demonstrated for the first time that human dermal fibroblast-derived EVs contain eccDNA.
• Observed variability in eccDNA detection across different bioinformatic pipelines and biological replicates.
• Characterized eccDNA ranging from 306 to 28,958 base pairs, with uneven chromosomal distribution and gene fragments in nearly half of the identified eccDNAs.

Conclusions:

• Human dermal fibroblast EVs are carriers of eccDNA, suggesting a role in intercellular cargo transfer.
• Methodological variability impacts eccDNA identification, highlighting the need for standardized approaches.
• This research provides a foundation for exploring eccDNA in EVs concerning skin aging and dermal cell biogenesis.