Abstract
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.
Highlights
May occur through the disruption of important RNA-protein or RNA-RNA interactions that are essential for RNA func
Our results indicate that antisense activity can be significantly enhanced through the use of high affinity 2‘ sugar modifications provided they are equipped with RNase H-sensitive deoxy gaps of the appropriate length
2“Modified Antisense Inhibitors of Ha-ras structures in Ha-ras mRNA in the codon 12 region [50, 56], Ability to directRNaseH cleavage of a complementary we extended the hybridization experiments described above RNA by 2”O-methyl deoxy gap oligonucleotides was deterto include a larger Ha-ras target which has previously been mined in vitro using HeLa nuclear extracts
Summary
A series of oligodeoxynucleotidephos- due to thethermodynamic costs of disrupting RNA secondary phorothioates ranging in length between 5 and 25 bases were structure prior to antisense oligonucleotide binding. Since tested for antisense activity and selectivity for the Ha-ras previous reports have claimed the existence of secondary sequence containing a G + T transversion at codon 12 [46]. Based on the sequence of the mutant selective 17-mer, a series of chimeric phosphorothioate 2”O-methyl oligonucle-. Uniform phosphorothioate linkage was included in these oligonucleotides to ensure stability against serum andintracellular nucleases.’. These oligonucleotides, along with a non-chimeric 2”O-methyl 17-mer Uniform phosphorothioate linkage was included in these oligonucleotides to ensure stability against serum andintracellular nucleases.’ These oligonucleotides, along with a non-chimeric 2”O-methyl 17-mer
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