Strategy for plasmid-based gene delivery in cells through ligand-receptor interaction.

V. SIGOT, D.J. ARNDT-JOVIN, T.M. JOVIN

Estocolmo

Taller; III EuroGeneDrug Project Meeting Report; 2005

The main objective as partner IV in this project was to develop a strategy for plasmid based gene delivery into cells through ligand-receptor interaction and study this interaction using fluorescence microscopy.In our system we used the technique referred as Bioplex (biological complex) that was developed by Partner I in which we attach a biological function, a biotinylated peptide nucleic acid (biotin-PNA), to a nucleic acid via sequence-specific hybridization.We used a biotin-PNA that specifically binds to a unique target site in pEGFP C1 plasmid, without interfering the ability to express the GFP’s reporter gene. Specific hybridization insures that plasmids are added in exact amounts to target cells through ligand receptor interaction, the extent of the gene delivery can be followed by “in vivo” expression of the reporter or potential therapeutic gene carried in the plasmid.We were particularly interested in receptor mediated internalization of these biotin-PNA:plasmid hybrids as a complex with highly luminiscent and stable streptavidin coated Quantum Dots (QD). Therefore we functionalized the QD with the biotinylated ligand and the biotin-PNA:plasmid complex to follow the receptor mediated internalization and intracellular distribution.In the previous results described in the last report we demonstrated the specificity of biotin-PNA binding to its cognate sequence on pEGFP-C1 plasmid using fluorescent labeled streptavidin and fluorescent labeled PNA. Specificity was also demonstrated by band shift assay on agarose gels with DNA fragments containing the target site for PNA.Then we demonstrated the specific binding of streptavidin coated QD to this hybrids complex on biotinylated magnetic beads and the retarded migration of the whole QD-biotin-PNA:plasmid complex on 0.8% agarose gels.