Oligonucleotide N3'--&gt;P5' phosphoramidates as antisense agents

Gryaznov, S. M.

doi:10.1093/nar/24.8.1508

Cited by 120 publications

(52 citation statements)

References 32 publications

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“…1) in d(AnTnGnAnCn)rU are resistant to chemical and nuclease degradation, a requirement for an effective therapeutic (20,(34)(35)(36). The results show that the oxygen to amino and 2Ј OH to 2Ј H functional group modifications (Fig.…”

Section: Discussionmentioning

confidence: 90%

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

Testa

Gryaznov

Turner

1999

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

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Binding enhancement by tertiary interactions is a strategy that takes advantage of the higher order folding of functionally important RNAs to bind short nucleic acid-based compounds tightly and more specifically than possible by simple base pairing. For example, tertiary interactions enhance binding of specific hexamers to a group I intron ribozyme from the opportunistic pathogen Pneumocystis carinii by 1,000-to 100,000-fold relative to binding by only base pairing. One such hexamer, d(AnTnGnAnCn)rU, contains an N3 3 P5 phosphoramidate deoxysugar-phosphate backbone (n) that is resistant to chemical and enzymatic decay. Here, it is shown that this hexamer is also a suicide inhibitor of the intron's self-splicing reaction in vitro. The hexamer is ligated in trans to the 3 exon of the precursor, producing dead-end products. At 4 mM Mg 2؉ , the fraction of trans-spliced product is greater than normally spliced product at hexamer concentrations as low as 200 nM. This provides an additional level of specificity for compounds that can exploit the catalytic potential of complexes with RNA targets.Most human therapeutics have been discovered by screening natural products. Synthetic organic chemistry has made it possible to synthesize such natural products and derivatives thereof in large quantities, thus broadening the range of compounds that can be used clinically (1-3). Synthetic methodology coupled with the outpouring of protein structural information has also allowed rational design of completely new therapeutic compounds (4,5). Similarly, the recent explosion in nucleic acid sequence information is providing a knowledge base for structure-based targeting of RNA. The first generation of such therapeutics consists of antisense nucleic acids that bind mRNA through Watson-Crick base pairing and thereby regulate translation (6, 7). Because of the long sequences employed, typically 15-20 nucleotides, potential disadvantages include high cost of synthesis (8) and lack of specificity (9). Cost of synthesis can be reduced and specificity increased by designing short antisense agents whose binding to RNA targets is enhanced by tertiary interactions (10, 11). Here we show that one such hexanucleotide can also be a suicide inhibitor (12, 13) of RNA function. This provides an additional design principle for increasing specificity of compounds that can exploit the catalytic potential of complexes with RNA targets.Our model system is a large-subunit ribosomal RNA (rRNA) precursor from the opportunistic pathogen Pneumocystis carinii, which is a common cause of death in immunocompromised patients (14, 15). The rRNA precursor contains a group I self-splicing intron (10, 16) that provides a potential therapeutic target (16, 17) because self-splicing is required for assembly of active ribosomes (18). Hexamers that mimic this intron's 5Ј exon can bind to the catalytic core of a ribozyme derived from the intron as much as 100,000-fold more tightly than expected if the hexamers bound by simple base pairing (10). Much of this bindi...

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Section: Discussionmentioning

confidence: 90%

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

Testa

Gryaznov

Turner

1999

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

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“…These results are consistent with (i) the increase in triplex stability of np oligonucleotides as previously described, and (ii) the resistance toward nucleases observed with np oligonucleotides under our experimental conditions (data not shown; ref. 38). Also, nuclear proteins have been shown to bind non-sequencespecifically to po oligonucleotides (36).…”

Section: Resultsmentioning

confidence: 99%

“…Also, nuclear proteins have been shown to bind non-sequencespecifically to po oligonucleotides (36). Preliminary experiments have shown that cellular proteins bind np oligomers less efficiently than po oligonucleotides (38). This may lead to the better availability of np than of po oligomers.…”

Section: Resultsmentioning

confidence: 99%

Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target

Giovannangéli

Diviacco

Labrousse

et al. 1997

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

157

120

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The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of doublehelical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.Oligonucleotides can be designed to bind to double-stranded DNA at oligopurine⅐oligopyrimidine sites where they form a local triple-helical structure (see refs. 1 and 2 for reviews). This provides a general approach to the sequence-specific recognition and targeting of double-stranded DNA for both basic research and therapeutic applications. However, the description of triplex-mediated effects in cell cultures is still rare. Inhibition of gene expression by triplex-forming oligonucleotides has been demonstrated on plasmid targets transiently transfected into living cells (3-7). When a triplex was preformed with the target in vitro and then the complex was transfected into mammalian cells, inhibition of gene expression was observed. We have previously shown that transcription of the gene coding for the ␣-subunit of the interleukin 2 receptor was inhibited when cells were first transfected with a reporter plasmid in the presence of a psoralenoligonucleotide conjugate and then irradiated (3), or when transfected cells were incubated with an acridineoligonucleotide conjugate without any irradiation (8, 9). Mutations have been detected on plasmid vectors after irradiation of transfected cells in the presence of psoralenoligonucleotide conjugates (10). Even in the absence of irradiation a low level of mutation was detected and was attributed to transcription-coupled repair (11). Location of the mutation sites was as it was expected on the basis of triplex formation. Detection of the DNA mutations is a very sensitive method to demonstrate triplex formation, but it does not allow quantitative analysis of the amount of triplex formed within cells. Using dimethyl sulfate footprinting ...

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“…However, the use of oligos is still hindered by several critical problems, including an instability to nuclease, sequence nonspecificity, and inadequate cellular uptake (10,11). A variety of chemically modified oligos, including phosphorothioate and methylphosphonate oligos, have been developed as a measure to augment stability against nucleases.…”

Section: Introductionmentioning

confidence: 99%

Prevention of CCl4-induced liver cirrhosis by ribbon antisense to transforming growth factor-β1

Doh¹,

Jung²,

Moon³

et al. 2008

Int J Mol Med

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Abstract. Transforming growth factor-ß1 (TGF-ß1) is an important mediator of tissue fibrosis, including liver cirrhosis. Ribbon-type antisense oligonucleotide to TGF-ß1 (TGF-ß1 RiAS) was designed and combined with cationic peptide derived from the nuclear localization signal of human immunodeficiency virus-1 Tat protein for enhanced cellular uptake. When Hepa1c1c7 cells were transfected with TGF-ß1 RiAS, the level of TGF-ß1 mRNA was reduced by >70%. TGF-ß1 RiAS, mismatched RiAS, and normal saline were each injected into mice via the tail vein, beginning the week after intraperitoneal CCl 4 injection and continuing for 7 weeks, in order to determine whether TGF-ß1 RiAS prevents the fibrotic changes induced by the CCl 4 injection. After 8 weeks of the experiment, all of the mice treated with TGF-ß1 RiAS survived, compared to 50% of the control group and 65% of the mismatched RiAS-treated group. Upon examining the biochemical effects on the liver, TGF-ß1 mRNA levels were reduced significantly only in the TGF-ß1 RiAS-treated group. Immunohistochemical studies showed a reduced accumulation of collagen and α-smooth muscle actin. Our experimental results suggest that ribbon antisense to TGF-ß1, with efficient uptake, effectively blocks the expression of TGF-ß1 and prevents fibrosis of the liver.

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Oligonucleotide N3'-->P5' phosphoramidates as antisense agents

Cited by 120 publications

References 32 publications

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target

Prevention of CCl4-induced liver cirrhosis by ribbon antisense to transforming growth factor-β1

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