Optimizing the isolation and characterization of cytosolic DNA from human cell lines

• Maryia Hrynashka, Applied Molecular Biology & Biotechnology, University of Delaware

• Brigette Romero, Applies Molecular Biology and Biotechnology, University of Delaware
• Mona Batish, Applied Molecular Biology and Biotechnology, University of Delaware

Cytosolic DNA refers to DNA fragments that have leaked out from the nucleus into the cytoplasm, typically due to cellular damage. This damage can result from infections, cancer, or certain drugs, particularly DNA damage repair (DDR) inhibitor drugs, which cause double-stranded DNA breaks leading to chromosome-free DNA. Once broken off, these DNA segments migrate into the cytoplasm, where they are known as cytosolic DNA and serve as indicators of several cancers. The presence and quantity of cytosolic DNA can be used to measure the effectiveness of DDR inhibitor drugs and serve as reliable pharmacodynamic markers. However, obtaining pure cytosolic DNA samples free of mitochondrial and nuclear DNA contaminants remains a challenge. Therefore, the primary goal of this project is to optimize an existing protocol for isolating cytosolic DNA from T98G, a glioblastoma cell line. Additionally, we aimed to visualize and quantify cytosolic DNA using single-molecule fluorescence in-situ hybridization (smFISH). To obtain pure cytosolic DNA, cells were lysed and then treated with proteinase K and RNase A to eliminate protein and RNA contaminants in the sample. To evaluate the purity of the sample, PCR was performed using primers targeting cytosolic DNA, a promoter, and an mRNA such as GAPDH. For the smFISH protocol, we adapted existing methods to image cytosolic DNA. Untreated cells and cells treated with 10 nM cytarabine for 16 hours were grown on glass coverslips, then fixed and permeabilized. Cells were hybridized with primary probes (20-25 oligos of 60 nucleotides in length) containing tails at the 5′ and 3′ ends. After washing, cells were hybridized with tail probes linked to a fluorophore (Texas Red). Additionally, DNase and RNase treatments were performed to ensure that any signals in the acquired images truly represent cytosolic DNA. The ability to obtain pure cytosolic DNA samples and to visualize and quantify cytosolic DNA will provide a novel basis for the analysis of DDR inhibitor drugs and inform clinical trials. This research has the potential to enhance our understanding of cellular damage mechanisms and improve cancer treatment strategies.