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J Biol Chem, Vol. 274, Issue 31, 21783-21789, July 30, 1999
From the Lineberger Comprehensive Cancer Center and Department of
Pharmacology, University of North Carolina,
Chapel Hill, North Carolina 27599
A novel, positive read-out assay that quantifies
only sequence-specific nuclear activity of antisense oligonucleotides
was used to evaluate morpholino and 2'-O-methyl
sugar-phosphate oligonucleotides. The assay is based on modification of
the splicing pathway of human
Antisense Oligonucleotides with Different Backbones
MODIFICATION OF SPLICING PATHWAYS AND EFFICACY OF UPTAKE
-globin pre-mRNA. In addition,
scrape-loading of cells with oligonucleotides allows the separate
assessment of intracellular antisense activity of the oligonucleotides
and their ability to penetrate the cell membrane barrier. The results
show that, with scrape-loading, the morpholino oligonucleotides were
approximately 3-fold more effective in their intrinsic antisense
activity than alternating phosphodiester/phosphorothioate
2'-O-methyl-oligoribonucleotides and 6-9- and almost
200-fold more effective than the exclusively phosphorothioate and
phosphodiester derivatives, respectively. The morpholino
oligonucleotides were over 20-fold more effective than the
phosphorothioate 2'-O-methyl-oligoribonucleotides in free
uptake from the culture media. The antisense activity of the morpholino
oligonucleotides was detectable not only in monolayer HeLa cells but
also in suspension K562 cells. Time course experiments suggest that
both the free uptake and efflux of morpholino oligonucleotides are slow.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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