PRECONCENTRATION AND SEPARATION OF ANTISENSE OLIGONUCLEOTIDES BY ON-COLUMN ISOTACHOPHORESIS AND CAPILLARY-ELECTROPHORESIS IN POLYMER-FILLEDCAPILLARIES

Small, single-stranded, chemically modified oligonucleotides, compleme ntary to a specific gene section, commonly referred to as antisense co mpounds, are being investigated as potential therapeutic drugs. A numb er of modified oligonucleotides, in particular phosphorothioates, are in clinical development. Shorter fragments are found as metabolic prod ucts. Isotachophoresis (ITP) allows the introduction of large, diluted sample plugs into the separation capillary. In this work, ITP and cap illary electrophoresis (CE) in polymer solutions were successfully cou pled in a single capillary in a commercial instrument to increase sens itivity with UV detection and to shorten the time for sample pretreatm ent. It was shown that ITP-CE can be used as a preconcentration and cl ean-up method for phosphodiester- and phosphorothioate-containing samp les. Up to 3 mu L sample could be injected into the capillary without significantly disturbing the separation performance. ITP-CE of phospho diesters directly out of salt- and protein-containing samples could be demonstrated. For phosphorothioates in serum samples an additional sa mple clean-up was necessary, due to oligonucleotide-protein binding. A n optimized replaceable polymer solution was developed to increase the separation performance for heterogeneous phosphorothioates. A dextran -based sieving medium showed a good separation performance in ITP-CE o f phosphorothioates. A concentration detection limit of 8.10(-9) mol/L for the 20-mer phosphorothioate ISIS5132, isolated from rat serum, wa s found.