Molecular organization of protein-DNA complexes for cell-targeted DNA delivery

Multifunctional proteins are interesting candidates for nonviral gene trans fer to and expression in their target cells. Since at difference of viral v ectors, the performance of these vehicles depends on their functional optim isation, a better comprehension of the molecular organisation within protei n-DNA complexes would be of great help in reaching their full delivery pote ntial. In this work, we have characterised an RGD-tagged, cell-targeted mul tifunctional beta -galactosidase carrying a poly-lysine-based DNA-binding d omain. 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 cond ensed 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 expr ession level of cell-delivered genes and integrin-binding specificity are b oth achieved at 0.02 mug of DNA per mug of protein, indicative of influence s of the packaged nucleic acid on the interaction between filled vehicles a nd the receptors of target cells. (C) 2000 Academic Press.