Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression.
We have used a previously described 17-mer phosphorothioate (Monia, B.P., Johnston, J.F., Ecker, D. J., Zounes, M.A., Lima, W.F., and Freier, S.M. (1992) J. Biol. Chem. 267, 19954-19962) for structure-function analysis of 2'-sugar modifications including 2'-O-methyl, 2'-O-propyl, 2'-O-pentyl, and 2'-fluoro. These modifications were analyzed for hybridization affinity to complementary RNA and for antisense activity against the Ha-ras oncogene in cells using a highly sensitive transactivation reporter gene system. Hybridization analysis demonstrated that all of the 2'-modified oligonucleotides hybridized with greater affinity to RNA than an unmodified 2'-deoxy oligonucleotide with the rank order of affinity being 2'-fluoro > 2'-O-methyl > 2'-O-propyl > 2'-O-pentyl > 2'-deoxy. Evaluation of antisense activities of uniformly 2'-modified oligonucleotides revealed that these compounds were completely ineffective in inhibiting Ha-ras gene expression. Activity was restored if the compound contained a stretch of at least five 2'-deoxy residues. This minimum deoxy length correlated perfectly with the minimum length required for efficient RNase H activation in vitro using partially purified mammalian RNase H enzyme. These chimeric 2'-modified/deoxy phosphorothioates displayed greater antisense potencies in inhibiting Ha-ras gene expression, compared with the unmodified uniform deoxy phosphorothioate. Furthermore, antisense potency correlated directly with affinity of a given 2' modification for it's complementary RNA. These results demonstrate the importance of target affinity in the action of antisense oligonucleotides and of RNase H as a mechanism by which these compounds exert their effects.
Department of Molecular Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008.
B P
BP
Monia
E A
EA
Lesnik
C
C
Gonzalez
W F
WF
Lima
D
D
McGee
C J
CJ
Guinosso
A M
AM
Kawasaki
P D
PD
Cook
S M
SM
Freier
D001482:Base Composition; D001483:Base Sequence; D002678:Chimera; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D015870:Gene Expression / Q000187:drug effects*; D011905:Genes, ras / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D016914:Ribonuclease H; D013539:Simian virus 40 / Q000235:genetics; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation
D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D016914:Ribonuclease H