Nuclease Resistance and Antisense Activity of Modified Oligonucleotides Targeted to Ha-ras

Brett P Monia,Joseph F Johnston,Henri Sasmor,Lendell L Cummins

doi:10.1074/jbc.271.24.14533

Brett P Monia, Joseph F Johnston + Show 2 more

Open Access

https://doi.org/10.1074/jbc.271.24.14533

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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.

Highlights

Fects in cells, was conferred to phosphodiester oligonucleotides by incorporation of appropriate 2؅-alkoxy sugar modifications
Relative nuclease stability of the modified oligonucleotides did not contribute significantly to relative antisense activity, since activity was assessed at short times following oligonucleotide treatment and the oligonucleotides were synthesized as stable uniform phosphorothioates
We have used this 17-mer sequence to test the effects of replacing PϭS backbone linkages with PϭO linkages on both nuclease stability in vitro and antisense activity in cells

Summary

Introduction

Fects in cells, was conferred to phosphodiester oligonucleotides by incorporation of appropriate 2؅-alkoxy sugar modifications. High concentrations of phosphorothioates have been shown to competitively inhibit a variety of nucleases and polymerases [6, 7, 12,13,14], interact with and potentially abrogate the activity of heparin-binding growth factors [13, 15], induce immune stimulatory effects in rodents [13, 16], cause complement activation and hypotension in monkeys, and induce clotting abnormalities in monkeys as a result of direct interactions with thrombin [17, 18] These potential limitations have not proven to be problematic in clinical trials to date, evaluation of novel oligonucleotide mod-. To alleviate the problem of nucleolytic degradation, chemical deoxy substituent with 2Ј-O-methyl and 2Ј-O-allyl modificamodifications of the natural phosphodiester backbone have tions has been reported to increase oligonucleotide stability been introduced into oligonucleotides to increase their stability toward various nucleases under cell-free conditions [22, 23]

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