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.
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 alpha-globin mRNA, inhibited non-specifically beta-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of beta-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 beta-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.
Larrouy
B
B
INSERM U 386, Laboratoire de Biophysique Moléculaire, Université Bordeaux II, France.
Boiziau
C
C
Sproat
B
B
Toulmé
J J
JJ
D000478:Alkylation; D001483:Base Sequence; D002474:Cell-Free System; D005914:Globins / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009843:Oligoribonucleotides / Q000737:chemistry*; D014176:Protein Biosynthesis; D016372:RNA, Antisense / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry*; D016914:Ribonuclease H / Q000378:metabolism*
D009843:Oligoribonucleotides; D016372:RNA, Antisense; D012333:RNA, Messenger; D005914:Globins; D016914:Ribonuclease H