title: Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras.
abstract: We have previously described structure-activity studies on a 17-mer uniform phosphorothioate antisense sequence targeted to human Ha-ras. In an effort to further improve the pharmacological properties of antisense oligonucleotides, structure-activity studies on this 17-mer sequence were expanded to examine both the effects of replacing phosphorothioate backbone linkages with phosphodiester linkages and the effects of incorporating various 2'-sugar modifications into phosphorothioate and phosphodiester oligonucleotides on oligonucleotide stability against nucleases in vitro and on antisense activity in cells. Replacement of three or more phosphorothioate linkages with phosphodiester linkages greatly compromised both nuclease resistance and antisense activity, and these effects correlated directly with the number of phosphodiester linkages incorporated into the oligonucleotide. However, substantial nuclease resistance, sufficient for obtaining potent antisense effects in cells, was conferred to phosphodiester oligonucleotides by incorporation of appropriate 2'-alkoxy sugar modifications. Nuclease stability and antisense activity imparted by these sugar modifications in phosphodiester backbones correlated with the size of the 2'-alkoxy substituent (pentoxy > propoxy > methoxy > deoxy). Furthermore, antisense activity mediated by oligonucleotides that exhibit partial resistance to nucleolytic degradation was dependent on both oligonucleotide concentration and the duration of oligonucleotide treatment.
authors.affiliation: Department of Molecular Pharmacology and Division of Medicinal Chemistry, Isis Pharmaceuticals, Carlsbad, California 92008, USA.
authors.forename: B P
authors.initials: BP
authors.lastname: Monia
authors.forename: J F
authors.initials: JF
authors.lastname: Johnston
authors.forename: H
authors.initials: H
authors.lastname: Sasmor
authors.forename: L L
authors.initials: LL
authors.lastname: Cummins
mesh_terms: D000970:Antineoplastic Agents / Q000633:toxicity*; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera; D015195:Drug Design; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D011905:Genes, ras; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000378:metabolism* / Q000633:toxicity*; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013873:Thionucleotides; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms
chemical_list: D000970:Antineoplastic Agents; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013873:Thionucleotides; D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I