A Method to Evaluate a Propidium Iodide Association with Oligonucleotides and Oligonucleotide-Cationic Lipid Interactions

Magda      Przybylo; Tomasz      Borowik; Andrzej      Okruszek; Marek      Langner

Abstract

Complex molecular ensembles are frequently considered an element of new pharmacological formulations. This is especially evident in the therapies based on genetic information. In order to obtain an effective drug, it is necessary to associate a nucleic acid molecule with the components to ensure the desired aggregate structure and properties. To evaluate the progress of the supromolecular aggregate formation a range of methodologies and techniques are needed to test the quality and uniformity of the formulations. In this paper we propose a procedure which measures the association of a small molecule with nucleic acid using propidium iodide and oligonucleotides as an example. To measure propidium iodide binding constant the oligonucleotide was covalently labeled with fluorescein and then the changes in fluorescence resonance energy transfer (FRET) were determined and handled according to the acceptor-donor titration methodology. The calculated binding constants were in a good agreement with the values published previously. The developed method was then used to evaluate the extent of an oligonucleotide association with the lipid aggregates. It was found that two populations of oligonucleotides are present in all lipid samples studied. The fraction of oligonucleotides associated with liposomes rises with the increase of a cationic lipid content, reaching the constant value when the fraction of cationic lipid exceeded 20 mol%. Energy transfer data combined with these obtained in quenching experiments show that the orientation of the oligonucleotide associated with a lipid bilayer depends on the amount of surface charge.

Keywords: Supramolecular aggregates, antisense oligonucleotides, gene therapy, lipid bilayer, fluorescence