Quantitation of intracellular concentration of a delivered morpholino oligomer by capillary electrophoresis-laser- induced fluorescence: correlation with upregulation of luciferase gene expression.
Antisense oligonucleotides have shown great promise over the past several years as viable drugs to combat various forms of cancer and viral diseases. However, quantitative detection to monitor cellular association is difficult using conventional methods such as radiolabeling of the oligonucleotide or fluorescence confocal microscopy. In this paper quantitation of intracellular concentration of the morpholino oligonucleotide is investigated using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). HeLa cells, which produce luciferase as the antisense oligomer enters the cell, were scrape-loaded with varying concentrations of the morpholino antisense. The intracellular antisense concentration measured by CE-LIF was found to correlate with those obtained with the cellular functional assay based on upregulation of luciferase. Intracellular concentrations of the antisense were found to be in the range of 6 to 29 nmol/g total cell protein, depending on the amounts that were scrape-loaded. To our best knowledge, this is the first reported quantitative correlation between delivered antisense concentration in a cell extract and the subsequent antisense upregulation of gene expression.
McKeon
J
J
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
Cho
M J
MJ
Khaledi
M G
MG
D002462:Cell Membrane / Q000502:physiology; D016503:Drug Delivery Systems; D019075:Electrophoresis, Capillary / Q000379:methods*; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D015870:Gene Expression; D006367:HeLa Cells / Q000378:metabolism*; D006801:Humans; D007834:Lasers; D008156:Luciferases / Q000235:genetics*; D008560:Membrane Fluidity; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000032:analysis*; D004364:Pharmaceutical Preparations / Q000032:analysis*; D015854:Up-Regulation
D005452:Fluoresceins; D005456:Fluorescent Dyes; D016376:Oligonucleotides, Antisense; D004364:Pharmaceutical Preparations; C024098:6-carboxyfluorescein; D008156:Luciferases