150. Cooperative strand invasion of super-coiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Top of pageAbstract Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical, and in the future possibly also medical applications. Among suggested functions are as specific anchor sequence for binding biologically active peptides to plasmids in a sequence specific manner via strand invasion. Such complexes, referred to as Bioplex, has already been used to enhance non viral gene transfer in vitro. Here we present a method to quantify the specific binding of a PNA to super coiled DNA by labeling the PNA with the fluorophore thiazole orange (TO). Using this method we have studied how the hybridization of multiple PNAs and PNA-peptide chimeras to sequences on the same DNA strand in a super coiled plasmid are effected by the distance between the binding sites (BSs). The kinetics of the strand invasion into a set of plasmids with different distances between the BSs indicate that the optimal space between two sites are two bases. At this distance the remaining cooperative effects are overriding the negative steric hindrance of a neighboring PNA-peptide. In addition we show increased binding kinetics when using two PNA binding to overlapping sites on the opposite strands without the use of chemically modified nucleobases in the PNA molecules.