RNase H is responsible for the non-specific inhibition of in vitro translation by 2′-O-alkyl chimeric oligonucleotides: High affinity or selectivity, a dilemma to design antisense oligomers

Abstract

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 [Larrouy et al. (1992), Gene, 121, 189-194], we demonstrated that ODN1, a phosphodiester 15mer targeted to the AUG initiation region of α-globin mRNA, inhibited non-specifically β-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of β-globin mRNA. Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogue of ODN1, 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 β-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. © 1995 Oxford University Press.