Analysis of oligonucleotide binding, internalization, and intracellular trafficking utilizing a novel radiolabeled crosslinker.
Although antisense oligonucleotides have been widely used to inhibit gene expression, their mechanism of entry into cells and their site of action are still in some doubt. In this report, we describe a novel technique for kinetically analyzing oligonucleotide association with living cells as well as intracellular compartmentalization. The technique utilizes a photoactivatable, radiolabeled crosslinker, the Denny-Jaffe reagent. Oligonucleotides containing pendant amine groups were conjugated to this reagent, added to HL60 cells in culture, and photocrosslinked to associated proteins, which were analyzed electrophoretically. We find that several proteins are labeled, predominantly a 75 kD one that appears to be membrane-associated. Our results suggest that the majority of intracellular oligonucleotide is associated in vesicles with the same protein to which it bound on the cell surface, but only a small percentage of non-protein-bound cytosolic oligonucleotide can be detected. Additionally, oligonucleotides are readily accumulated by nuclei, and by treating whole nuclei, a unique set of nuclear binding proteins is detected.
Geselowitz
D A
DA
Clinical Pharmacology Branch, NCI, NIH, Bethesda, Maryland.
Neckers
L M
LM
D001386:Azides; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013388:Succinimides
D001386:Azides; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D013388:Succinimides; C077904:Denny-Jaffe reagent