No abstract

Reverse transcription is one step of the retroviral development which can be inhibited by antisense oligonucleotides complementary to the RNA template. 2'-O-Alkyl oligoribonucleotides are of interest due to their nuclease resistance, and to the high stability of the hybrids they form with RNA. Oligonucleotides, either fully or partly modified with 2'-O-alkyl residues, were targeted to an RNA template to prevent cDNA synthesis by the Avian Myeloblastosis Virus reverse transcriptase (AMV RT). Fully-modified 2'-O-allyl 17mers were able to specifically block reverse transcription via an RNase H-independent mechanism, with efficiencies comparable to those observed with phosphodiester (PO) and phosphorothioate oligonucleotides. Sandwich 2'-O-alkyUIPO/2'-0-alkyl oligonucleotides, supposed to combine the properties of 2'-O-alkyl modifications (physical blocking of the RT) to those of the PO window (RNase H-mediated cleavage of the RNA) were quasi-stoichiometric inhibitors when adjacent to the primer, but remained without any effect when non-adjacent. They were not able to compete with the polymerase and inhibited reverse transcription only through RNase H-mediated cleavage of the target.

Ribonuclease H (RNase H) which recognizes and cleaves the RNA strand of mismatched RNA-DNA heteroduplexes can induce non-specific effects of antisense oligonucleotides. In a previous paper [Lar-rouy et a!. (1992), Gene, 121,[189][190][191][192][193][194], we demonstrated that ODN1, a phosphodiester 1 5mer targeted to the AUG initiation region of a-globin mRNA, inhibited non-specifically 3-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of f-globin mRNA. Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogue of ODNI, which limited RNase H activity on non-perfect hybrids. We report here that 2'-O-alkyl RNA-phosphodiester DNA sandwich analogues of ODN1, with the same phosphodiester window as MP-ODN2, are non-specific inhibitors of globin synthesis in wheat germ extract, whatever the substituent (methyl, allyl or butyl) on the 2'-OH. These sandwich oligomers induced the cleavage of non-target f-globin RNA sites, similarly to the unmodified parent oligomer ODN1. This is likely due to the increased affinity of 2'-O-alkyl-ODN2 chimeric oligomers for both fully and partly complementary RNA, compared to MP-ODN2. In contrast, the fully modified 2'-O-methyl analogue of ODN1 was a very effective and highly specific antisense sequence. This was ascribed to its inability (i) to induce RNA cleavage by RNase H and (ii) to physically prevent the elongation of the polypeptide chain.

Biochemical Society Transactionslikely efficacy of antisense oligodeoxynucleotides as chemotherapeutic agents. Two major problems remain. The first of these, as mentioned earlier, is the stability of the ODNs in cell systems. Our present experiments suggest that the half life of these is between 1 and 3 h, which is unlikely to be sufficient for a clinical preparation. The second concerns the efficiency of uptake into the cells which, while we have not yet quantified it, we believe is unlikely to be greater than 10% of the total amount of ODN administered. It will therefore be essential by suitable means (e.g. liposome encapsulation) [8] to increase this uptake.