Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake.
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 beta-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.
Lineberger Comprehensive Cancer Center and Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
G
G
Schmajuk
H
H
Sierakowska
R
R
Kole
D017398:Alternative Splicing / Q000187:drug effects*; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008745:Methylation; D009025:Morpholines; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology*; D012322:RNA Precursors / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D009025:Morpholines; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D005914:Globins