Noncoding DNA in Lipofection of HeLa Cells—A Few Insights

Abstract

In cationic carrier-mediated gene delivery, the disproportional relationship between the quantity of delivered DNA and the amount of encoded protein produced is a well-known phenomenon. The numerous intracellular barriers which need to be overcome by pDNA to reach the nucleoplasm play a major role in it. In contrast to what one would expect, a partial replacement of coding pDNA by noncoding DNA does not lead to a decrease in transfection efficiency. The mechanism underlying this observation is still unclear. Therefore, we investigated which constituents of the transfection process might contribute to this phenomenon. Our data reveal that the topology of the noncoding plasmid DNA plays a major role. Noncoding pDNA can be used only in a supercoiled form to replace coding pDNA in Lipofectamine lipoplexes, without a loss in transfection levels. When noncoding pDNA is linearized or partly digested, it diminishes the transfection potential of coding pDNA, as does noncoding salmon DNA. The difference in transfection efficiencies could not be attributed to diverse physicochemical characteristics of the Lipofectamine lipoplexes containing different types of noncoding DNA or to the extent of their internalization. At the level of endosomal release, however, nucleic acid release from the endosomal compartment proceeds faster when lipoplexes contain noncoding salmon DNA. Since the half-life of pDNA in the cytosol hardly exceeds 90 min, it is conceivable that prolonged release of coding pDNA from complexes carrying supercoiled noncoding pDNA may explain its positive effect on transfection, while this depot effect does not exist when noncoding salmon DNA is used.

Cell division responsive peptides for optimized plasmid DNA delivery: The mitotic window of opportunity?

“…Currently, cells are challenged with an excessive amount of pDNA to reach in vitro gene delivery. We and others, however, found that more than 90% of the originally administered pDNA can be replaced by noncoding pDNA, demonstrating the enormous potential to further optimize pDNA delivery and decrease the dose applied to cells [21,22]. In this research paper, we evaluated two strategies to increase the fraction of initially applied pDNA that reaches the nuclear interior of the cells.…”

Chondroitin Sulfate‐Coated DNA‐Nanoplexes Enhance Transfection Efficiency by Controlling Plasmid Release from Endosomes: A New Insight into Modulating Nonviral Gene Transfection

“…Notably, in cases of pDNA/PEI/CS and pDNA/PEI/CS‐CHO nanoplexes, the transfection efficiency markedly diminished and became nearly identical to that of pDNA/PEI‐based transfection experiment (Figure B). This demonstrates that controlled release of the nanoplexes from endolysosomal compartments is critical for improving transfection efficiency as the half‐life of pDNA in the cytosol rarely exceeds 90 min …”

“…Cationic polymer such as PEI can favor endosomal escape by “proton sponge effect” initiating the swelling of the endosomes . Recently, Symens et al reported that prolonged release of coding pDNA from nanoplexes by diluting the pDNA with noncoding DNA could favor transfection efficiency . Thus, tuning endosomal release of pDNA could have significant implication on transfection efficiency as this could prevent premature degradation of the DNA in cytosol.…”

“…Recently, new evidence suggests that fast release of plasmid may not be ideal for effective gene expression. Controlling the plasmid release rate from the endosome by simply adding noncoding DNA along with coding DNA and cationic polymer has shown to prolong DNA release, resulting in enhanced transfection efficiency . This controlled release could be due to the endosome destabilization resulting in pore formation that leaks nanoparticles without rupture of these organelles .…”

Characterizing the Non-Normal Distribution of Flow Cytometry Measurements from Transiently Expressed Constructs in Mammalian Cells

“…For example, the observed plateau that occurs for the shape parameter in the transfection concentration series (Fig. 4) may be indicative of a saturation effect on one or more of the mechanisms that mediate DNA delivery, e.g., escape from the endosomes, cytoplasmic concentration during mitosis, or nuclear shuttling activities 5,7,[9][10][11][12][13] . Alternatively, it might reflect a transition in the dominating source of noise, where stochastic expression effects begins to dominate over gene-delivery variance.…”