Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages

Marcus-Sekura, Carol J.; Woerner, Amy M.; Shinozuka, Kazuo; Zon, Gerald; Quinnan, Gerald V.

doi:10.1093/nar/15.14.5749

Cited by 171 publications

(72 citation statements)

References 63 publications

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“…Substitution of the phosphodiester backbone of native DNA with phosphorothioate moieties markedly augments resistance to nuclease digestion. 35,36 Phosphorothioates, however, have also been found to bind with high affinity to a large number of proteins 37,38 presumably the result of alterations in the presentation of negative charges. Without appropriate controls, sequence-independent effects caused by phosphorothioate chemistry can be mistakenly attributed to a true antisense effect.…”

Section: Discussionmentioning

confidence: 99%

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

Santiago

Atkins

Khachigian

1999

The American Journal of Pathology

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Smooth muscle cell (SMC) proliferation is a key event in renarrowing of blood vessels after balloon angioplasty. Mechanical injury imparted to the arterial wall in experimental models induces the expression of the immediate-early gene , egr-1. Egr-1 binds to and activates expression from the proximal promoters of multiple genes whose products can , in turn , influence the vascular response to injury. Here , we used antisense strategies in vitro to inhibit rat vascular SMC proliferation by directly targeting Egr-1. A series of phosphorothioate antisense oligonucleotides of 15 base length and complementary to various theoretically accessible regions within Egr-1 mRNA were synthesized and assessed for their ability to selectively inhibit SMC proliferation in an Egr-1-dependent manner. Western blot analysis revealed that two oligonucleotides , AS2 and E11 , inhibited Egr-1 synthesis in cells exposed to serum without affecting levels of the zinc finger protein Sp1. AS2 and E11 inhibited seruminducible [ 3 H]thymidine incorporation into DNA, as well as serum stimulation of total cell numbers. Sizematched phosphorothioate oligonucleotides with random , scrambled , sense or mismatch sequences failed to inhibit. Antisense Egr-1 inhibition was nontoxic and reversible. These oligonucleotides also inhibited SMC regrowth after mechanical injury in vitro. Egr-1 thus plays a key regulatory role in SMC proliferation and repair following injury. (Am J Pathol 1999, 155:897-905)

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Section: Discussionmentioning

confidence: 99%

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

Santiago

Atkins

Khachigian

1999

The American Journal of Pathology

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“…The optimal length of a PNA or PMO depends on achieving a balance between specificity and efficacy and is estimated to be about 15 to 25 bases (2, 14, 16, 21, 30). However, experimental evidence suggests that many factors, such as cellular uptake or invasion of mRNA secondary structure, influence efficacy and can tip the balance in favor of shorter antisense oligomers (13,15,19,23,31).Sequence-specific antibacterial drugs are a recent application of antisense technology (11,12,17,24,25,33). Nielsen and coworkers (13) have shown that PNA in the 9-to 12-mer range were more active inhibitors than larger oligomers in Escherichia coli.…”

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confidence: 99%

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confidence: 99%

Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition inEscherichia coli

Deere

Iversen

Geller

2005

Antimicrob Agents Chemother

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Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs. Conversely, in bacterial cell-free protein synthesis reactions, longer PMOs inhibited equally or more than shorter PMOs. Overlapping, isometric (10-base) PMOs complementary to the region around the start codon of luciferase inhibited to different extents in bacterial cell-free protein expression reactions. Including the anti-start codon in PMOs was not required for maximal inhibition. PMOs targeted to 5 nontranslated or 3 coding regions within luciferase mRNA did not inhibit, except for one PMO targeted to the ribosome-binding site. Inhibition of luciferase expression correlated negatively with the predicted secondary structure of mRNA regions targeted by PMO but did not correlate with C؉G content of targeted regions. The effects of PMO length and position were corroborated by using PMOs (6 to 20 bases) targeted to acpP, a gene required for viability. Because inhibition by PMOs of ϳ11 bases was unexpected based on previous results in eukaryotes, we tested an 11-base PMO in HeLa cells and reticulocyte cell-free protein synthesis reactions. The 11-base PMO significantly inhibited luciferase expression in HeLa cells, although less than did a 20-base PMO. In reticulocyte cell-free reactions, there was a trend toward more inhibition with longer PMOs. These studies indicate that strategies for designing PMOs are substantially different for prokaryotic than eukaryotic targets.Antisense drugs are short (10 to 25 bases) oligomers that mimic DNA or RNA and inhibit gene expression in a sequence-dependent manner (2, 6, 29, 30). They differ from native RNA or DNA in the chemical structure that links the four common bases.Most of the reported work on antisense drugs has been accomplished in eukaryotic systems. In eukaryotes, antisense compounds inhibit by two general mechanisms. Compounds such as phosphorothioates hybridize to mRNA and promote its degradation by RNase H (3, 5, 28). Other compounds, such as peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomer (PMO), hybridize to specific mRNA and block translation by an RNase H-independent mechanism (3, 28).The most effective region for targeting PNA and PMO is the 5Ј untranslated region and initiation codon of mRNA (3,10,14,15,18,20,28,30,32). The optimal length of a PNA or PMO depends on achieving a balance between specificity and efficacy and is estimated to be about 15 to 25 bases (2, 14, 16, 21, 30). However, experimental evidence suggests that many factors, such as cellular uptake or invasion of mRNA secondary structure, influence efficacy and can tip the balance in favor of shorter antisense oligomers (13,15,19,23,31).Sequence-specific antibacterial drugs are a recent application of antisense technology (11,12,17,24,25,33). Nielsen and cowork...

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“…All oligodeoxyribonucleotides (oligonucleotides) were synthesized on an Applied Biosystems 380B DNA synthesizer. Phosphorothioates containing sulfur at each internucleotide linkage were prepared by sulfurization of the corresponding oligonucleotide hydrogen phosphonates by using Applied Biosystems recommended synthesis cycles and adaptations of previously described methods (36)(37)(38).…”

mentioning

confidence: 99%

Mouse Mos protooncogene product is present and functions during oogenesis.

Paules

Buccione

Moschel

et al. 1989

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

157

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Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages

Cited by 171 publications

References 63 publications

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition inEscherichia coli

Mouse Mos protooncogene product is present and functions during oogenesis.

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