Quantitative nuclear and cytoplasmic localization of antisense oligonucleotides by capillary electrophoresis with laser-induced fluorescence detection.
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.
McKeon
J
J
Department of Chemistry, North Carolina State University, Raleigh 27695, USA.
Khaledi
M G
MG
D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000648:ultrastructure*; D003593:Cytoplasm / Q000648:ultrastructure; D016373:DNA, Antisense / Q000032:analysis*; D003911:Dextrans; D019075:Electrophoresis, Capillary / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D013050:Spectrometry, Fluorescence / Q000379:methods; D013873:Thionucleotides
D016373:DNA, Antisense; D003911:Dextrans; D013873:Thionucleotides; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate