RNase H-mediated inhibition of translation by antisense oligodeoxyribo-nucleotides: use of backbone modification to improve specificity

RNase H is responsible for the non-specific inhibition ofin vitrotranslation by 2′-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers

“…Antisense oligonucleotides 15 nt long are expected to lead to specific inhibitory effects (29). However, RNase H which cleaves the RNA target of RNA-DNA heteroduplexes, can act on mismatched hybrids (15,30 (18,19). Due to the problem raised by stereo-isomers in methylphosphonate derivatives it was of interest to check whether similar properties could be displayed by other kinds of sandwich molecules.…”

“…After electroblotting the nylon membrane was probed with 32P 5' end-labeled oligonucleotide complementary to nt 3-19 of the ,-globin RNA. Hybridization was performed as described previously (19).…”

“…Therefore, RNase H can cleave non-target RNA sites exhibiting partial complementarity to the antisense oligonucleotide (15), leading to non-specific inhibition in some eukaryotic cells (16,17). In order to reduce the cleavage of non-perfect hybrids, we and others previously used methylphosphonate sandwich oligomers which led to an increased specificity of the antisense chimeric oligomer compared to the unmodified one (18)(19)(20). We had previously shown that a l5mer, with a central window of five phosphodiesters inserted between two methylphosphonate flanks, targeted to the initiation codon region of the rabbit a-globin mRNA, displayed some selectivity in the inhibition of in vitro globin synthesis, despite the presence, on the f,-globin mRNA, of nine sites sharing more than 60% homology with the primary a-target site (19 phodiester DNA chimeras.…”

“…In order to reduce the cleavage of non-perfect hybrids, we and others previously used methylphosphonate sandwich oligomers which led to an increased specificity of the antisense chimeric oligomer compared to the unmodified one (18)(19)(20). We had previously shown that a l5mer, with a central window of five phosphodiesters inserted between two methylphosphonate flanks, targeted to the initiation codon region of the rabbit a-globin mRNA, displayed some selectivity in the inhibition of in vitro globin synthesis, despite the presence, on the f,-globin mRNA, of nine sites sharing more than 60% homology with the primary a-target site (19 phodiester DNA chimeras. We demonstrate here that selectivity is not restored with such oligonucleotides which bind much more strongly to the RNA than methylphosphonate-phosphodiester oligomers.…”

Restoration of antibiotic susceptibility in fluoroquinolone-resistant Escherichia coli by targeting acrB with antisense phosphorothioate oligonucleotide encapsulated in novel anion liposome

“…Furthermore, in contrast to the steric-blocker oligonucleotides, RNase H-dependent oligonucleotides, such as phosphorothioate oligonucleotides, can inhibit protein expression when targeted to widely separated areas in the coding region. [18][19][20] Mechanisms in fluoroquinolone resistance of E. coli fall into two principal categories: alterations in drug targets (for example DNA gyrase or topoisomerase IV) [21][22][23][24][25][26] and decreased cellular accumulation of quinolones. The latter involves the major and constitutively expressed multi-drug efflux pump, AcrAB-TolC.…”