Sorption of oligonucleotides on positively charged lipid membranes

It has been demonstrated that oligonucleotides, small segments of DNA, can prevent synthesis of undesirable proteins by blocking the transmission of genetic information.

In order to produce a specific protein, the gene is first transcribed from the double stranded DNA into a single strand of messenger RNA (m-RNA). Subsequently, information on m-RNA is translated into the molecular structure of protein. To block the transmission, small segments of single stranded DNA, typically 15-20 nucleotides long, are used to “turn-off the gene”.

The effectiveness of this method, and the use of oligonucleotides as drugs, is limited by several major obstacles. One of the is the transport of oligonucleotides into the cytoplasm or nucleus where the disruption of gene expression must occur. Our project is concerned with the problem of their sorption to membrane as the first step of the internalization process.

Internalization of oligonucleotides is a complex process. It is now well established that increased lipophilicity of oligonucleotides by conjugation with lipophilic groups and cholesterol improves their efficiency even though the mechanism of transport is not understood. We are pursuing this approach.

There are no experimental data in the literature indicating relationships between the membrane binding characteristics of oligonucleotides, the oligonucleotide length, the molecular structure of bases, electric charge and the charge distribution within the oligonucleotide molecule and the structure of positively charged lipids. These are topics to be addressed in the research project.

The aim of the experiments is to obtain membrane partition coefficients of the oligonucleotides and then to estimate the Gibbs free energy of binding (?G), a major thermodynamic parameter determining oligonucleotide binding to membranes. Knowledge of the relationship between ?G and the molecular structure of the oligonucleotides is essential for the understanding of oligonucleotide internalization in cells.

?G will be related to (a) the molecular structure of the nucleotides (length, the number of monomer units and the units of negative charge), and (b) the positive electric charge on the lipid membrane (the surface density of membrane electric charge) and the molecular structure of positively charged lipids.

The surface concentrations of oligonucleotides (in numbers/area) will be obtained from the surface electric charge of spherical membrane lipid vesicles (about ?m in diameter), also called liposomes, to which the oligonucleotides adsorb. The density of electric charge on the liposome surface is determined from the drift velocity of liposomes in applied electric field. This quantity is measured directly using electrophoretic mobility analyzer. The advantage of this approach is that the electrophoretic mobility provides information on the electric potential at the surface of the liposome. Information on electrostatic potential is typically not available from conventional studies of binding using labeled oligonucleotides. Our experimental methodology has a definite advantage over the conventional membrane partition studies because it enables us (1) to study the electrostatic interactions between the membrane surface and the oligonucleotides in more direct way and (2) to obtain intrinsic (and not apparent, as in other studies) binding parameters for oligonucleotides.