Cellular Defense Mechanisms Show Stronger Response to Linear DNA Compared to Circular Plasmid [RNA-seq]

Low transfection efficiency poses a significant challenge to experimental success, making the improvement of gene-carrying plasmid delivery and expression essential across various research fields. The stability of transfected vector DNA or mRNA significantly impacts transgene expression, as the innate immune system may recognize plasmid DNA as foreign and initiate a degradation response. Currently, it is unclear whether RNA transcribed from plasmids can activate RNA sensors, affecting transfection efficiency. This study employed RNA sequencing to analyze cellular responses to various circular and linear DNAs across five cell lines—HEK293T, HCT116, HeLa, L02, and NCM460—revealing that the innate immune response is a primary contributor to low transfection efficiency. Additionally, we used ChIP-seq to detect H3K27ac histone marks to explore whether the immune response triggered by plasmid transfection is regulated by epigenetic mechanisms. Finally, we performed ChIP assays for IRF7 and p-IRF3 in HeLa cells transfected with the linear pcDNA3.1-neo plasmid to demonstrate that these two transcription factors can indeed bind to the RTV genes. Overall design: We performed gene expression profiling analysis using data obtained from RNA-seq of HEK293T, HCT116, HeLa, L02, and NCM460 cells transfected with an empty vector, pcDNA3.1-neo plasmid, linear pcDNA3.1-neo plasmid, pre-gRNA plasmid, or linear pre-gRNA plasmid. Additionally, we incorporated RNA sequencing data from L02 and HCT116 cells transfected with linear pcDNA3.1-neo plasmid and linear pre-gRNA plasmid treated with PNK or AP.