Molecular Characterization of Extrachromosomal MicroDNAs in Human Cancer Cell Lines Using Synthetic Minicircles

Recent studies in the Dutta Lab have identified microDNA, a novel form of extrachromosomal circular DNA (eccDNA) that is found within normal and cancerous vertebrate tissues and cell lines (Science, 2012, 336:86). These microDNA differ from previously identified eccDNA in that they are short (< 400 bp in length) and are preferentially derived from unique non‐repetitive regions of the genome that have a high GC content, high gene density, high exon density and promoters with activating chromatin modifications. Currently, little is known about the generation and stability of microDNA within cells. In order to characterize these properties of microDNAs, we employed synthetic minicircle constructs that can be introduced into cells and model the behavior of endogenous microDNAs, but with improved detection. Here, we examine the stability of microDNA by measuring the half‐life of these synthetic microDNAs in various immortalized cancer cell lines using quantitative Real Time Polymerase Chain Reaction and Southern blotting techniques. Additionally, to investigate the molecular mechanism of microDNA stability within the cell, we altered various cellular processes using chemical inhibitors and measured the effect on microDNA half‐life. Combined, these experiments characterize important features of this novel nucleic acid species and provide the basis for further exploration into the function of microDNA. This work was supported by the National Institutes of Health (ROI CA60499 and POI CA104160 to A.D.), the Ovarian Cancer Research Fund (290250 to L.W.D.), and the Charles Henry Leach, II, Foundation College Science Scholars Award (to D.R.P.).