Quantitative nuclear and cytoplasmic localization of antisense oligonucleotides by capillary electrophoresis with laser-induced fluorescence detection.

Abstract

We demonstrate the use of simple extraction procedures to separate nuclear and cytoplasmic material from cell extracts, which have been scrape-loaded with a 2-O-methyl phosphorothioate antisense oligonucleotide. Separation and quantitation of the fluorescein-labeled antisense and the flourescein isothiocyanate (FITC)-dextran (molecular weight 40000) as an internal standard is done using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). The bulky FITC-dextran is unable to penetrate the nuclear membrane thereby making it a quantitative indicator of any overlap between the nuclear and cytoplasmic materials during separation of the two phases. Using this procedure, the fluorescein-labeled phosphorothioate oligomer was quantitated at 4.1 x 10(-13) and 3.4x 10(-14) mol antisense/microg-total cellular protein in the nuclear and cytoplasmic extracts respectively following scrape-load delivery of the phosphorothioate to a batch of confluent HeLa cells at a concentration of 0.5 microM (5 x 10(-10) total moles of oligomer). Additionally, gene expression was monitored by measurement of the luciferase reporter protein activity. Scrape-load, spontaneous and liposomal delivery were investigated and compared for subcellular distribution of the oligomer and subsequent gene expression.