RNASE-H IS RESPONSIBLE FOR THE NONSPECIFIC INHIBITION OF IN-VITRO TRANSLATION BY 2'-O-ALKYL CHIMERIC OLIGONUCLEOTIDES - HIGH-AFFINITY OR SELECTIVITY, A DILEMMA TO DESIGN ANTISENSE OLIGOMERS

Ribonuclease H (RNase H) which recognizes and cleaves the RNA strand o f 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 15 mer targeted to the AUG initiation region of alpha-globin mRNA, inhibi ted non-specifically P-globin synthesis in wheat germ extract due to R Nase H-mediated cleavage of beta-globin mRNA, Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogu e of ODN1, which limited RNase H activity on non-perfect hybrids, We r eport here that 2'-O-alkyl RNA-phosphodiester DNA sandwich analogues o f ODN1, with the same phosphodiester window as MP-ODN2, are non-specif ic inhibitors of globin synthesis in wheat germ extract, whatever the substituent (methyl, allyl or butyl) on the 2'-OH, These sandwich olig omers induced the cleavage of non-target beta-globin RNA sites, simila rly to the unmodified parent oligomer ODN1, This is likely due to the increased affinity of 2'-O-alkyl-ODN2 chimeric oligomers for both full y and partly complementary RNA, compared to MP-ODN2, In contrast, the fully modified 2'-O-methyl analogue of ODN1 was a very effective and h ighly specific antisense sequence, This was ascribed to its inability (i) to induce RNA cleavage by RNase H and (ii) to physically prevent t he elongation of the polypeptide chain.