Digoxigenin-labeled phosphorothioate oligonucleotides: a new tool for the study of cellular uptake.
The mechanisms and intracellular pathways by which many oligonucleotide analogs enter cells to exert the desired antisense effects are not fully understood and remain a matter of debate. In this study, we describe the synthesis of 5'-digoxigenin-labeled phosphorothioate oligonucleotides and show their use to examine intracellular oligonucleotide distribution within Epstein-Barr virus-transformed B cells. Comparison of digoxigenin-labeled and fluorescein-labeled oligonucleotide distribution shows the same intracellular fate, suggesting that digoxigenin modification does not interfere with intracellular routing. Double immunofluorescence studied by conventional fluorescence and confocal microscopy with antibodies to the labeling molecule and to lysosome-associated membrane protein indicate that oligonucleotides mainly accumulate in the lysosomal compartment. Digoxigenin labeling offers an alternative to study oligonucleotide uptake and distribution by immunoelectron microscopy. Two different approaches have been studied: immunogold labeling in heavily fixed and resin-embedded cells and immunogold labeling in lightly fixed and cryoultramicrotomy processed cells. The results confirm the major lysosomal accumulation of digoxigenin-labeled oligonucleotides and demonstrate that the antigenic capacity of digoxigenin is not damaged by any of the procedures used. Therefore, the conjugation of the functionalized digoxigenin molecule at the 5' end of phosphorothioate oligonucleotides provides a new tool in the study of oligonucleotide uptake and intracellular distribution at both cellular and ultrastructural levels.
Tarrasón
G
G
Laboratorio Bioinvestigación, Merck Farma y Química, S.A., Barcelona, Spain.
Bellido
D
D
Eritja
R
R
Vilaró
S
S
Piulats
J
J
D001402:B-Lymphocytes; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D002472:Cell Transformation, Viral; D002851:Chromatography, High Pressure Liquid; D004076:Digoxigenin; D005452:Fluoresceins; D004854:Herpesvirus 4, Human; D006801:Humans; D007202:Indicators and Reagents; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000032:analysis / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D013329:Structure-Activity Relationship; D013873:Thionucleotides
D005452:Fluoresceins; D007202:Indicators and Reagents; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D004076:Digoxigenin