BACKGROUND: Plasmid DNA (pDNA) dissociation from polyamine gene vectors after cellular uptake has not been well characterized. A more detailed understanding of this process could lead to more efficient gene transfer agents. Since RNA is present in the cytoplasm at high concentrations and due to its structural similarity to DNA, we were interested in its conceivable interaction with polyamine gene vectors. METHODS: In a first set of experiments gene vectors were incubated in cell lysate and pDNA release was investigated by Southern blot analysis with or without RNase A pretreatment and by confocal laser scanning microscopy. Further, interaction of polyamine gene vectors with RNA was investigated by fluorescence quenching assay. These methods were complemented by a functionality assay using isolated nuclei. RESULTS: The incubation of gene vectors with cell lysate resulted in the dissociation of pDNA from the complexes. This effect was abolished when the cell lysate was pretreated with RNase A. The addition of RNA in the absence of cell lysate led also to a dissociation of pDNA. This process commenced instantaneously after the addition of RNA as analyzed by fluorescence quenching. When gene vectors were incubated in cell lysate containing isolated nuclei, the dissociation of pDNA from the polyamine gene vectors occurred preferentially extranuclearally as confirmed by confocal laser scanning microscopy. These results were further corroborated in a functional assay. CONCLUSIONS: These data suggest that RNA induces pDNA dissociation from the polyamine gene vectors. Furthermore, this process apparently occurs in the cytoplasm before the gene vectors enter the nucleus. Copyright 2006 John Wiley & Sons, Ltd.
BACKGROUND: Plasmid DNA (pDNA) dissociation from polyamine gene vectors after cellular uptake has not been well characterized. A more detailed understanding of this process could lead to more efficient gene transfer agents. Since RNA is present in the cytoplasm at high concentrations and due to its structural similarity to DNA, we were interested in its conceivable interaction with polyamine gene vectors. METHODS: In a first set of experiments gene vectors were incubated in cell lysate and pDNA release was investigated by Southern blot analysis with or without RNase A pretreatment and by confocal laser scanning microscopy. Further, interaction of polyamine gene vectors with RNA was investigated by fluorescence quenching assay. These methods were complemented by a functionality assay using isolated nuclei. RESULTS: The incubation of gene vectors with cell lysate resulted in the dissociation of pDNA from the complexes. This effect was abolished when the cell lysate was pretreated with RNase A. The addition of RNA in the absence of cell lysate led also to a dissociation of pDNA. This process commenced instantaneously after the addition of RNA as analyzed by fluorescence quenching. When gene vectors were incubated in cell lysate containing isolated nuclei, the dissociation of pDNA from the polyamine gene vectors occurred preferentially extranuclearally as confirmed by confocal laser scanning microscopy. These results were further corroborated in a functional assay. CONCLUSIONS: These data suggest that RNA induces pDNA dissociation from the polyamine gene vectors. Furthermore, this process apparently occurs in the cytoplasm before the gene vectors enter the nucleus. Copyright 2006 John Wiley & Sons, Ltd.
Authors: Alex Moretti; Marianna Ponzo; Charles A Nicolette; Irina Y Tcherepanova; Andrea Biondi; Chiara F Magnani Journal: Front Immunol Date: 2022-06-09 Impact factor: 8.786