Direct electrochemical detection of oligonucleotide hybridization on poly(5-hydroxy-1,4-naphthoquinone- co-5-hydroxy-3-thioacetic acid-1,4-naphthoquinone) film.

We describe the construction of a new DNA-modified electrode based on an electroactive film. 5-Hydroxy-1,4-naphthoquinone is coelectrooxidized with 5-hydroxy-3-thioacetic acid-1,4-naphthoquinone to give a copolymer, presenting both electroactive and chemically reactive groups. The carboxylic function acts as a precursor for the covalent grafting of ODN probes while the quinone group acts as the transduction element of hybridization. Electrochemical detection was performed by differential pulse voltammetry in the electroactivity domain of the quinone group (i.e., at very low potentials, 0 to -0.8 V vs SCE). A very clear modification of the redox activity is observed between unmodified and probe-modified films and especially upon addition of target ODN.

Electrochemical method for entrapment of oligonucleotides in polymer-coated electrodes.

Oligonucleotides were incorporated into conducting films of poly(3, 4-ethylene dioxythiophene) by an electrochemical method that involves two steps. The electrode is first coated with the polymer film by electropolymerization followed by electrooxidation of the formed polymer in the presence of the oligonucleotide. Neutral water soluble polymers such as poly(vinylpyrrolidone) or poly(ethylene glycol) were added to the polymerizing medium resulting in higher incorporation yields of oligonucleotides. The results showed that a payload of about 1.5 nmol/cm2 can be achieved in an 8 micron thick film for an oligomer concentration of about 70 microM in the working solution. Various physical methods were used to analyze the surface of the polymer-coated electrodes. Results showed the presence of domains of varying sizes at the film surface, corresponding to the insertion of the hydrophilic polymer within the matrix of the conductive polymer. The release of entrapped oligonucleotides from the coated film exhibited a three-step profile: a "burst" period during the first 5 min followed by an intermediate and a very slow release period lasting several days. Such polymer films could be exploited as useful reservoirs of biologically active substances for in vivo delivery to targeted tissues. For example, coated stents that can release antiproliferative agents such as antisense oligonucleotides at the site of balloon dilatation may be of interest in the treatment of postangioplasty restenosis.

High specific radioactivity labeling of oligonucleotides with 3H-succinimidyl propionate.

An easy and rapid method for tritium labeling of deprotected oligonucleotides is proposed. The method consists in performing the reaction of commercial 3H-succinimidyl propionate with a terminal amino group of the oligonucleotide in an organic medium. High specific radioactivity labeling can be achieved with minimal radiolysis during long term storage. The synthesis of the nonradioactive congener having an identical structure to the labeled compound is also described.

Cell binding, uptake and cytosolic partition of HIV anti-gag phosphodiester oligonucleotides 3'-linked to cholesterol derivatives in macrophages.

The purpose of this study is to evaluate the cell interactions of a new class of compounds composed of phosphodiester oligonucleotides linked to the cholesterol group at position 3, 7, or 22 of the steroid structure. The resulting conjugates were assessed for their capacity to bind, penetrate and partition in the cytoplasmic compartment of murine macrophages. The results showed that lipophilic conjugates bind to cells much faster (t(1/2) < or = 10 min) than do underivatized oligomers. Oligomers tethered to the cholesterol at positions 3 and 7 (PO-GEM-3-Chol and PO-GEM-7-Chol) interacted more efficiently with cell membranes and were better internalized than oligomers attached to the cholesterol moiety at position 22 (PO-GEM-22-Chol). The cytosolic fraction of internalized oligomers was studied by a digitonin-based membrane permeabilization method. The recovered fraction of oligomers that can freely diffuse from the cytosol was comparable for GEM-91, a phosphorothioate congener, and for PO-GEM-7-Chol (50-60% of the internalized oligomers), while that of PO-GEM-3-Chol was less (30% of the internalized oligomers) indicating a higher membrane affinity of the latter derivative as compared to the other investigated compounds. Membrane binding and cell internalization correlated well with the hydrophobicity of the conjugates as characterized by their partition coefficients in a water-octanol system. Due to their capacity of rapid binding and cytosolic partition in cells, cholesterol-derivatized oligonucleotides at position 3 or 7 of the steroid molecule appeared as good candidates for systemic delivery of anti-HIV antisense compounds.