Molecular organization of protein-DNA complexes for cell-targeted DNA delivery.
Multifunctional proteins are interesting candidates for nonviral gene transfer to and expression in their target cells. Since at difference of viral vectors, the performance of these vehicles depends on their functional optimisation, a better comprehension of the molecular organisation within protein-DNA complexes would be of great help in reaching their full delivery potential. In this work, we have characterised an RGD-tagged, cell-targeted multifunctional beta-galactosidase carrying a poly-lysine-based DNA-binding domain. In solution, the engineered enzyme spontaneously forms proteinaceous particles of between 20 and 40 nm in diameter that might contain around 10 molecules of enzymatically active protein. Plasmid DNA is efficiently condensed into these particles without modification of the shape, morphology or enzymatic activity, indicative of a comfortable molecular accommodation to the DNA-binding domains. Although the RGD peptide remains equally solvent-exposed and immunoreactive at different DNA-protein ratios, an optimal expression level of cell-delivered genes and integrin-binding specificity are both achieved at 0.02 microg of DNA per microg of protein, indicative of influences of the packaged nucleic acid on the interaction between filled vehicles and the receptors of target cells.
Arís
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Institut de Biologia Fonamental and Departament de Genètica and Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 08193, Spain.
Villaverde
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D004247:DNA / Q000008:administration & dosage* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015316:Genetic Therapy; D008854:Microscopy, Electron; D008958:Models, Molecular; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D001616:beta-Galactosidase