α-Anomeric DNA: β-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase

Bloch, Evelyne; Lavignon, Marc; Bertrand, Jean-Rémi; Pognan, François; Morvan, François; Malvy, Claude; Rayner, Bernard; Imbach, Jean‐Louis; Paoletti, Claude

doi:10.1016/0378-1119(88)90162-x

Cited by 29 publications

(5 citation statements)

References 15 publications

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“…This process was essentially sequence-independent and rested on the preferential binding of the modified oligomers to the retroviral enzyme compared with mammalian DNA polymerases. Synthesis of cDNA by MMLV RT was also blocked by complementary oligonucleotides through a direct competition with the primer (17,28). In this case, the competitor, an a-oligonucleotide analogue, and the primer had the same sequence, but the former could not prime reverse transcription-hence, the inhibition.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides.

Boiziau

Thuong

Toulmé

1992

Proc. Natl. Acad. Sci. U.S.A.

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We have demonstrated that the synthesis of cDNA by avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases can be prevented by oligonucleotides bound to the RNA template =100 nucleotides remote from the 3' end of the primer. The RNA was truncated at the level of the antisense oligonucleotide-RNA duplex during the reverse transcription. The key role played by the reverse transcriptase-associated RNase H activity in the inhibition process was shown by the use of (i) inhibitors of RNase H (NaF or dAMP), (it) Moloney murine leukemia virus reverse transcriptase devoid of RNase H activity, or (iii) a-analogues of oligomers that do not elicit RNase H-catalyzed RNA degradation. In all three cases the inhibitory effect was either reduced (NaF, dAMP) or totally abolished. However, an a-oligomer bound to the sequence immediately adjacent to the primerbinding site prevented reverse transcription. Therefore, initiation of polymerization can be blocked by means of an RNase H-independent mechanism, whereas arrest of a growing cDNA strand can be achieved only by an oligonucleotide mediating cleavage of the template RNA.nucleotide inhibited syncytia formation, human immunodeficiency virus protein synthesis, or RT activity was not elucidated.We anticipated that the production of cDNA by retroviral polymerase could be inhibited by an oligonucleotide bound to the RNA downstream from the primer: the RT molecule traveling on the template would be blocked by the hybrid formed between the RNA and the antisense oligonucleotide ( Fig. 1). In a preliminary report we showed that an unmodified 17-mer, indeed, arrested cDNA synthesis by AMV RT (13). We present here a detailed analysis of the effect of antisense oligonucleotides on DNA polymerization by AMV and MMLV RTs. We have been able to demonstrate that the RNase H activity ofAMV and MMLV enzymes was involved in the process because a truncated RNA template was produced. a-Oligomers that are nuclease-resistant analogues of oligonucleotides (14,15) Since the use of synthetic oligonucleotides to inhibit the in vitro development of Rous sarcoma virus (5), the potential therapeutic interest of the antisense strategy has been recognized (6). The emergence of AIDS and the need for different approaches to control retroviruses have led to numerous studies in the field. The antiviral efficiency of both unmodified and modified antisense oligomers has been tested (7-12). Successful results have been reported with oligomers targeted to different sites of viral RNA or mRNA. However, in most cases, the mechanism by which the antisense oligo-MATERIALS AND METHODS Oligonucleotides. Unmodified ,8-and a-oligodeoxynucleotides listed in Table 1 were synthesized either on a Pharmacia or on a Biosearch automatic synthesizer. According to previous studies a-oligomers were designed to bind in a parallel orientation with respect to the RNA strand (16,17). Oligonucleotides linked at their 5' end to 2-methoxy-6-chloro-9-aminoacridine through a pentamethylene linker were prepared...

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Section: Discussionmentioning

confidence: 99%

Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides.

Boiziau

Thuong

Toulmé

1992

Proc. Natl. Acad. Sci. U.S.A.

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“…RNase H displays low sequence specificity, and it is presumed that it recognizes some general structural features of the heteroduplex, such as sugar-phosphate backbone (Hogreffe et al, 1990). Of note is that heteroduplexes formed by modified oligonucleotides containing an uncharged internucleotide linkage such as methylphosphonate, phosphotriester, and dephosphono linkage (Uhlmann & Peyman, 1990) or containing modified sugar such as 2′-Omethylribose (Inoue et al, 1987) or R-anomeric nucleoside (Bloch et al, 1988) are not substrates for RNase H. Therefore, it may be of interest that all CDU-modified oligonucleotides 2-7 RNA-DNA heteroduplexes with a 400-600 bases poly rA template were found to be substrate for RNase H. This observation is in agreement with the present knowledge on RNase H substrate specificity since CDU-oligonucleotides, although containing a modified base, retain an unchanged, charged backbone. Additionally, the efficacy of poly rA template digestion seems independent from T m of the duplex formed with CDU-oligonucleotide despite the finding that the T m of duplexes with CDUoligonucleotides varied from 15 to 29 °C (Fulcrand-El .…”

Section: Discussionmentioning

confidence: 99%

Carboranyl Oligonucleotides. 3. Biochemical Properties of Oligonucleotides Containing 5-(o-Carboranyl-1-yl)-2‘-deoxyuridine^,

et al. 1996

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Boronated oligonucleotides are potential candidates for boron neutron capture therapy, antisense technology, and as tools in molecular biology. The biological properties of dodecathymidylic acids containing one or more 5-(o-carboran-1-yl)-2'-deoxyuridine residues at different locations within the oligonucleotide chain were studied. 5-(o-Carboran-1-yl)-2'-deoxyuridine containing oligonucleotides manifested marked increased lipophilicity and resistance to 3'- or 5'-phosphodiesterases compared to the corresponding unmodified oligomer. They were substrates for T4 polynucleotide kinase and primers for Escherichia coli polymerase I and human immunodeficiency virus type 1 reverse transcriptase but not for human DNA polymerase alpha and beta. They also formed heteroduplexes that were substrates for E. coli RNase H, an essential property for antisense technology. These studies indicate that the carboranyl-containing oligonucleotides have desirable properties that need to be exploited further in the design of novel biopharmaceuticals.

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“…Previous reports have already shown that oligonucleotide analogues can be used to interfere with cDNA synthesis. In the case of oligomers 1 2 comprising ot-anomers of nucleoside units, which cannot be elongated by reverse transcriptase, inhibition was obtained through competition with the primer [16,17]. It was also reported that phosphorothiate derivatives could prevent HIV development in cultured cells by a sequence-independent mechanism involving, at least in part, the binding of these modified oligomers to the viral reverse transcriptase [18].…”

Section: Resultsmentioning

confidence: 99%

Blockage of AM V reverse transcriptase by antisense oligodeoxynucleotides

1990

FEBS Letters

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α-Anomeric DNA: β-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase

Cited by 29 publications

References 15 publications

Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides.

Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides.

Carboranyl Oligonucleotides. 3. Biochemical Properties of Oligonucleotides Containing 5-(o-Carboranyl-1-yl)-2‘-deoxyuridine^,

Blockage of AM V reverse transcriptase by antisense oligodeoxynucleotides

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α-Anomeric DNA: β-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase

Cited by 29 publications

References 15 publications

Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides.

Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides.

Carboranyl Oligonucleotides. 3. Biochemical Properties of Oligonucleotides Containing 5-(o-Carboranyl-1-yl)-2‘-deoxyuridine,

Blockage of AM V reverse transcriptase by antisense oligodeoxynucleotides

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Carboranyl Oligonucleotides. 3. Biochemical Properties of Oligonucleotides Containing 5-(o-Carboranyl-1-yl)-2‘-deoxyuridine^,