Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylpbosphonates in a cell-free system

Maher, L. James; Dolnick, Bruce J.

doi:10.1093/nar/16.8.3341

Cited by 115 publications

(44 citation statements)

References 47 publications

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“…The chance of a sequence of 5 bases occurring at random is therefore 45 or 1 in 1024. Since there are approximately 9200-9700 bases in the HIV genome, a given sequence of 5 bases may, on a random basis, occur nine times. Actually a computer search reveals a frequency of occurrence in the HIV genome of all possible tetramers ranging from 0 (CGCG) to 149 (AAAA).…”

Section: Discussionmentioning

confidence: 99%

“…Antisense oligodeoxynucleotides and their analogues have been used as tools for inhibiting viral replication (1-3), for blocking splicing and translation of mRNA (4,5), and for regulating specific gene expression (6,7). It has been found that an oligonucleotide complementary to a segment of a viral genome or an mRNA derived therefrom may interfere with the expression of that genetic segment by hybridization competition (1,2).…”

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confidence: 99%

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Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues.

Agrawal¹,

Ikeuchi²,

Sun³

et al. 1989

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

196

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Antisense oligodeoxynucleotides, both the phosphorothioate analogues and unmodified oligomers of the same sequence, inhibit replication and expression of human immunodeficiency virus already growing in tissue cultures of MOLT-3 cells with much greater efficacy than do mismatched ("random") oligomers and homooligomers of the same length and with the same internucleotide modification. This preferential inhibitory effect is elicited in as short a time as 4-24 hr postinfection. Likewise, antisense oligomers exhibit greater inhibitory effects on human immunodeficiency virus in chronically infected cells than do mismatched oligomers and homooligomers. Phosphorothioate antisense oligomers are up to 100 times more potent than unmodified oligomers of the same sequence in these inhibitory assays. These results, in major respects, confirm and extend those recently published by Matsukura et al. [Matsukura, M., Zon, G., Shinozuka, K., Robert-Guroff, M., Shimada, T., Stein, C. A., Mitsuza, H., Wong-Staal, F., Cohen, J. S. & Broder, S. (1989) Proc. Nati. Acad. Sci. USA 86,[4244][4245][4246][4247][4248]. They also point out the importance of computer analysis of sequences thought to be random but that in reality contain significant areas of likely hybridization, either to the viral genome or to the complementary DNA strand synthesized from it. They thus reinforce the concept that specific base pairing is a crucial feature of oligonucleotide inhibition of human immunodeficiency virus.Antisense oligodeoxynucleotides and their analogues have been used as tools for inhibiting viral replication (1-3), for blocking splicing and translation of mRNA (4, 5), and for regulating specific gene expression (6, 7). It has been found that an oligonucleotide complementary to a segment of a viral genome or an mRNA derived therefrom may interfere with the expression of that genetic segment by hybridization competition (1,2).In earlier reports, we demonstrated that replication of human immunodeficiency virus (HIV) could be inhibited by normal phosphodiester oligodeoxynucleotide sequences complementary to HIV RNA (4, 8). However, relatively short half-lives of normal oligonucleotides (9) in serum and in cells due to the presence of nucleases, and possibly the low permeability of these charged molecules into normal cells, limit their potential usefulness in vivo. To overcome this limitation, we and others (10-15) have studied internucleotide phosphate backbone-modified oligonucleotides, such as methylphosphonates (3, 10) as well as phosphorothioates and various phosphoramidates (11,12), for their antiviral activities against HIV.Here we report both the inhibition of HIV replication and the effect on cell growth of MOLT-3 cells that have been infected just 24 or 48 hr before addition of oligomers and also the effect on MOLT-3 cells chronically infected with HIV that were mixed with uninfected cells in the presence of oligodeoxynucleotides and their phosphorothioate analogues. MATERIALS AND METHODSOligonucleotide Synthesis. Oligonucleotides we...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues.

Agrawal¹,

Ikeuchi²,

Sun³

et al. 1989

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

196

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“…In particular, by extending the methylphosphonate sequences at each end of the molecule we may enhance cell uptake of the oligodeoxynucleotide in addition to protecting the molecule from exonucleases (Miller et al, 1981). In addition, by reducing the length of the internal phosphodiester sequence we may reduce the target for endonucleolytic degradation while retaining the ability of the molecule to direct ribonuclease-H cleavage of mRNA at the site of hybridisation (Donis-Keller, 1979), an anti-sense mechanism of sequence specific protein synthesis inhibition not exhibited by homogeneous methylphosphonate oligonucleotide analogues (Maher & Dolnick, 1988). We envisage that by reducing the number of phosphodiester linkages to the minimum required for ribonuclease-H activity it may be possible to target a specific site on the mRNA (Shibahara et al, 1987) and hence inhibit expression of a gene carrying a single point mutation at that site, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…The oligonucleotides, in the fully protected, trityl-on, controlled pore glass support-bound form, were deprotected following the procedure for methylphosphonate oligonucleotide analogues (Maher & Dolnick, 1988 N-ras anti-sense chimeric oligodeoxynucleotides…”

Section: Synthesis Of Oligodeoxynucleotidesmentioning

confidence: 99%

Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

Tidd

Warenius²

1989

Br J Cancer

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Summary Under certain circumstances sequence-specific inhibition of gene expression may be achieved in intact cells using exogenous anti-sense oligodeoxynucleotides. The efficacy of this approach to investigating gene function is limited in part by the rapid serum nuclease mediated degradation of oligodeoxynucleotides in culture media. In order to determine the relative contributions of 3'-exonuclease, 5'-exonuclease and endonuclease activity in fetal calf serum to oligodeoxynucleotide destruction, we have tested chimeric N-ras anti-sense sequence molecules protected against exonuclease attack with terminal methylphosphonate diester linkages. An 18-mer with two methylphosphonate diester linkages at the 3'-terminus, a 20-mer with two methylphosphonate diester groups at both ends, and the 16-mer 3'-methylphosphonate monoester components of their respective piperidine hydrolysates were totally resistant to venom phosphodiesterase, whereas the 16-mer 3'-hydroxyl components of the hydrolysates were rapidly degraded. Both the chimeric oligodeoxynucleotides and 3'-methylphosphonate monoesters were considerably more stable than normal 3'-hydroxyl oligodeoxynucleotides at 37'C in McCoy's 5A medium containing 15% heat inactivated fetal calf serum. Typically 20-30% of the former (initial concentration 10-100 jM) remained intact at 20 h as compared to the latter which were 88-100% degraded in 4 h and undetectable at 20 h. We conclude that a 3'-phosphodiesterase activity is a predominant nuclease responsible for oligodeoxynucleotide degradation by fetal calf serum, and that for cell culture studies, significant protection of oligodeoxynucleotides may be achieved by incorporating 3'-terminal methylphosphonate diester or even monoester end groups.

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“…Several modifications, introduced into oligomers to reduce their sensitivity to nucleases, impaired their ability to elicit RNase H activity. This is the case with methylphosphonates (3,4), phosphoramidates (5), 2'-O-alkyl RNA (6) and alpha analogues (4,7,8). Composite oligonucleotides made of differently modified (or unmodified) parts were prepared that aimed at combining different properties within a single molecule (9)(10)(11).…”

Section: Introductionmentioning

confidence: 98%

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

Larrouy¹,

Boiziau²,

Sproat

et al. 1995

Nucl Acids Res

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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 [Lar-rouy et a!. (1992), Gene, 121,[189][190][191][192][193][194], we demonstrated that ODN1, a phosphodiester 1 5mer targeted to the AUG initiation region of a-globin mRNA, inhibited non-specifically 3-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of f-globin mRNA. Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogue of ODNI, 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 f-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.

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Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylpbosphonates in a cell-free system

Cited by 115 publications

References 47 publications

Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues.

Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues.

Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

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

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