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PubMed1997

Pattern and kinetics of cytokine production following administration of phosphorothioate oligonucleotides in mice.

Phosphorothioate oligonucleotides with certain sequences or structure motifs can stimulate the immune system. We administered to mice a 27-mer phosphorothioate oligonucleotide (sequence 5'-TCG TCG CTG TCT CCG CTT CTT CTT GCC-3'), which has previously been shown to cause splenomegaly and hypergamma-globulinemia on in vivo administration in mice, and studied the pattern and kinetics of cytokine production at both the splenic mRNA and serum protein levels. Following i.p. administration of 50 mg/kg of oligonucleotide, significant increases in the splenic mRNA levels of IL-6, IL-12p40, IL-1 beta, and IL-1Ra and serum levels of IL-6, IL-12, MIP-1 beta, and MCP-1 were observed. In contrast, no significant differences in splenic mRNA levels of IL-2, IL-4, IL-5, IL-9, IL-13, IL-15, IFN-gamma, or MIF or serum levels of IL-2, IL-4, IL-5, IL-10, IFN-gamma, or GM-CSF were detected. The induction of IL-12 secretion was dependent on the sequence and dose of the oligonucleotides. One oligonucleotide (sequence 5'-GAG AAC GCT CGA CCT TCG AT-3') induced a high level of IL-12 secretion even at 5 mg/kg, whereas another oligonucleotide (sequence 5'-CTC TGC CAC CCA TCT CTC TCC TTC T-3') did not induce significant IL-12 secretion even at 50 mg/kg. IL-12 secretion induced by various doses of oligonucleotide has the same kinetics but differs in magnitude. These studies show a distinct pattern and kinetics of cytokine production following oligonucleotide administration and further demonstrate that cytokine induction is not a general property of phosphorothioate oligonucleotides but is dependent on the sequence and dose of the oligonucleotides.

Animals↗
PubMed1997

Stereodependent inhibition of plasminogen activator inhibitor type 1 by phosphorothioate oligonucleotides: proof of sequence specificity in cell culture and in vivo rat experiments.

Hexadecadeoxyribonucleotides complementary to a fragment of human PAI-1 mRNA located upstream of the start codon and their phosphorothioate analogs were studied in cultured HUVECs as sequence-dependent inhibitors of PAI-1 expression. The activity of the random mixture of diastereomers of phosphorothioate hexadecanucleotide PS-16H has been compared with that of isosequential, stereoregular [All-Sp] and [All-Rp] isomers. The highest inhibitory effect on PAI-1 synthesis was observed with the [All-Sp] diastereomer. Stereorandom phosphorothioate oligonucleotide PS-16R complementary to the same region of rat PAI-1 mRNA, when injected into tail vein of rats, substantially decreased the level of PAI-1 in blood plasma.

Animals↗
PubMed1998

Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies.

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

Antigens↗
PubMed1985

Evidence for sequence-specific conformational changes in DNA from the melting temperatures of DNA phosphorothioate derivatives.

Analogs of alternating purine-pyrimidine DNA polymers such as poly(dA-dT)-poly(dA-dT) can be made with phosphorothioate groups in the DNA backbone. A phosphorothioate diester at the 5'-purine-pyrimidine-3' step causes a significant lowering of the polymer's melting temperature compared to a phosphorothioate diester at the 5'-pyrimidine-purine-3' step. This may occur because sulfur substitution increases anionic charge density in the DNA minor groove and 5'-purine-pyrimidine-3' steps narrow the minor groove. The ability to modulate charge density in the DNA backbone via sulfur substitution should prove useful in studies of sequence-dependent conformational changes in DNA.

Crystallization↗
PubMed1987

An approach to the stereoselective synthesis of Sp-dinucleoside phosphorothioates using phosphotriester chemistry.

An approach to the stereoselective synthesis of Sp- dinucleoside phosphorothioates has been investigated which utilizes phosphotriester chemistry. The stereoselectivity of internucleotide bond formation between N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(S2-cyano-e thyl) phosphorothioate (3) and 3'-O-acetylthymidine has been studied using either mesitylenesulphonyl-5-(pyridin-2-yl)tetrazole (MSPy) or 1-mesitylenesulphonyl-3-nitro-1,2,4-triazole (MSNT) as the activating agent. The removal of the cyanoethyl group from the protected dinucleoside phosphorothioate has been studied, and conditions are reported which provide rapid deprotection without concomittant desulphurisation.

Benzene Derivatives↗
PubMed1988

Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide.

A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dye. The strand specificity of the reaction was investigated by employing the ethidium bromide mediated nicking reaction in the phosphorothioate-based oligonucleotide-directed mutagenesis method. The mutational efficiencies obtained were in the region of 64-89%, indicating that these restriction enzymes hydrolyse the phosphodiester bond at the cleavage site of the unsubstituted (+)strand.

Base Composition↗
PubMed1988

Autolytic processing of a phosphorothioate diester bond.

A small satellite RNA of tobacco ringspot virus replicates in tissues infected with tobacco ringspot virus and accumulates in virus capsids, forming virus-like particles. Previous research showed that multimeric forms of this satellite RNA have tandem repeats of the "monomeric" satellite RNA sequence of 359 or 360 nucleotide residues. The multimeric RNAs undergo autolytic processing at a specific CpA phosphodiester bond, the junction, to generate the monomeric RNA. We substituted phosphorothioate diester bonds for various sets of phosphodiester bonds, in dimeric and truncated forms of the satellite RNA. The degree of reduction in autolytic cleavage varied both with the sites of substitution and the size of the RNA molecules. Analyses of a product of the autolysis reaction suggest that one phosphorothioate diester bond most strongly interferes with processing, the one introduced at the CpA junction during its synthesis from adenosine-5'-0-(1-thiotriphosphate). However, extensive introduction of phosphorothioate diester bonds elsewhere in the molecule also decreased processing, possibly by altering conformation.

Base Sequence↗
PubMed1994

Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization.

We described a solid-supported desulfurization procedure allowing easy access to the sequence analysis of oligonucleotide phosphorothioates. The described method is based upon selective removal of the 2-cyanoethyl phosphate protecting groups, followed by iodine-promoted desulfurization of the resulting phosphorothioate diesters. Automatic oxidation of oligonucleotide phosphorothioates, anchored via an ester linkage to a standard solid support (LCAA/CPG), is combined with Maxam-Gilbert solid-support sequencing. The overall procedure allows rapid simultaneous sequence analysis of several oligonucleotide analogs.

Base Sequence↗
PubMed1994

Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner.

Animals↗
PubMed1994

Pyrimidine phosphorothioate oligonucleotides form triple-stranded helices and promote transcription inhibition.

The ability of phosphorothioate (POS) oligonucleotides to recognise and bind to homopurine-homopyrimidine DNA double-stranded sites via triple helix formation has been investigated. It has been found that the homologous pyrimidine POS sequences Y11-Si (i = 0, 1,2,3,4,10), which have been obtained by an increasing sulphur substitution in the sugar-phosphate backbone of d(CTTCCTCCTCT) (Y11), and the target hairpin duplex d(GAAGGAGGAGA-T4-TCTCCTCCTTC) (h26) can form stable triple helices, as indicated by PAGE, CD and UV melting experiments. The thermal stability of the triple helices depends on the number of POS linkages in the third Y11 strand, varying from 48 degrees C (Y11, with only phosphate groups, PO2) to 31 degrees C (Y11-S10 containing exclusively thioate groups). On average, a Tm depression of about 2 degrees C per POS linkage introduced in Y11 was observed. CD data indicate that the sulphurization of the third strand results in minimal changes of triple-stranded structures. The energetics of the triplex-to-hairpin plus single-strand transition has been determined by van't Hoff analyses of the melting curves. In free energy terms, the POS triplexes h26.Y11-Si are less stable than the normal PO2 h26.Y11 triplex by values between 2.7 and 5.4 kcal/mol, depending on the number of POS linkages contained in the third strand. Phosphorothioate oligonucleotides being resistant towards several nucleases offer an interesting choice as gene blockers in antisense strategy. Thus, their ability to inhibit transcription via triple helix formation has been examined in vitro. We found that triplex-forming POS oligonucleotides of 20 bases in length (with a cytosine contents of 45%), containing either 10% or 26% thioate groups, strongly repress the transcription activity of the bacteriophage T7 RNA polymerase at pH 6.9, when used in excess compared to the target (mol oligo/mol template = 125). The here reported data are useful for designing phosphorothioate oligonucleotides targeted to genomic DNA in antigene strategy.

Base Sequence↗
PubMed1995

Polyamine effects on purine-purine-pyrimidine triple helix formation by phosphodiester and phosphorothioate oligodeoxyribonucleotides.

Utilization of oligodeoxyribonucleotides to inhibit specific gene transcription in vivo (antigene strategy) requires the efficient formation of triple helices under physiological conditions. However, pyrimidine-motif triplexes are not favored at physiological pH, and physiological concentrations of potassium cations hamper purine-motif triplex formation. Here we investigated the effects of polyamines on promoting triplex formation by G/T-rich oligodeoxyribonucleotides containing either phosphodiester or a diastereomeric mixture of phosphorothioate linkages. Compared with Mg2+, equimolar concentrations of polyamines greatly facilitated purine-motif triplex formation with the following order of effectiveness: spermine > spermidine > putrescine. At low polyamine concentrations, phosphorothioate oligonucleotides were better at triplex formation than the corresponding phosphodiester oligonucleotides. Kinetic studies indicated that polyamines facilitated triplex formation by increasing the rate of oligonucleotide-duplex DNA association. However, triplex accumulation with either oligonucleotide was still low under physiological conditions (140 mM K+, 10 mM Mg2+, 1 mM spermine). The inhibitory effects of K+ could be partially overcome with high concentrations of Mg2+ or spermine, with phosphodiester oligonucleotides being better able to form triplexes than phosphorothioates under these conditions.

Base Sequence↗
PubMed1995

New efficient sulfurizing reagents for the preparation of oligodeoxyribonucleotide phosphorothioate analogues.

A set of new sulfurizing agents representing disulfides of arylsulfonic acids has been developed for the automated synthesis of phosphorothioate oligonucleotide analogues via the phosphoramidite method. These reagents, such as bis(benzenesulfonyl)disulfide, bis(p-toluenesulfonyl)disulfide, bis(p-methoxybenzensulfonyl)disulfide, and bis (p-chlorobenzenesulfonyl) disulfide, are easily prepared crystalline solid compounds. They are relatively inexpensive, easy to handle, and efficiently convert internucleotide cyanoethyl phosphite to the phosphorothioate triester within 1-2 min. The efficiency of phosphorothioate oligonucleotide synthesis with the use of these reagents is comparable to that of phosphodiester oligonucleotides.

Arylsulfonic Acids↗
PubMed1995

Binding of phosphorothioate oligodeoxynucleotides to basic fibroblast growth factor, recombinant soluble CD4, laminin and fibronectin is P-chirality independent.

Antisense oligodeoxynucleotides can selectively inhibit the expression of individual genes and thus have potential applications in anticancer and antiviral therapy. A critical prerequisite to their use as therapeutic agents is the understanding of their non-specific interactions with biological structures, e.g. proteins. In this study we examined the interactions of P-chiral phosphorothioate oligodeoxynucleotides with several proteins. The Rp- and Sp- diastereomers, and racemic machine-made mixtures, or M-oligodeoxynucleotides were used independently as competitors of the binding of a probe, phosphodiester oligodeoxynucleotide bearing a 5' alkylating moiety, to rsCD4, bFGF and laminin. These oligodeoxynucleotides were also used as competitors of the binding of a non-alkylating probe M-phosphorothioate oligodeoxynucleotide, 5'-32P-SdT18 to fibronectin. The average values of and quantitative estimates for the IC50 of competition and the constant of competition (Kc) of Rp-, Sp- and M-stereoisomers of several homo- and heteropolymer oligodeoxynucleotides were determined and compared. Surprisingly, in the proteins we studied, the values of IC50 and Kc for the Rp-, Sp- and M-oligodeoxynucleotides were essentially identical. Thus, the ability of the phosphorothioate oligodeoxynucleotides we employed, to bind to the proteins studied in this work, is virtually independent of P-chirality. Our results also imply that the role of the purine and pyrimidine bases in oligodeoxynucleotide-protein interactions, as well as the nature of the contact points (sulfur versus oxygen) between the oligomer and the protein, may be relatively unimportant.

Base Sequence↗
PubMed1997

Antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat alpha1(I) collagen gene promoter and inhibit transcription in cultured rat fibroblasts.

The rat alpha1(I) collagen promoter contains a unique polypurine-polypyrimidine sequence between -141 and -200 upstream of the transcription start site. The polypurine sequence from -171 to -200 (C2) is on the coding strand and the adjacent polypurine sequence from -141 to -170 (C1) is on the non-coding strand. Earlier we demonstrated triplex formation with a polypurine 30 nt parallel triplex-forming oligonucleotide (TFO) corresponding to C1 and inhibition of transcriptional activity of the rat alpha1(I) collagen promoter. In the present work we have tested triplex-forming abilities of shorter (18 nt) purine and pyrimidine TFOs in parallel and antiparallel orientation to the C1 purine sequence. Our results show that purine antiparallel TFOs formed triplexes with the highest binding affinities, while pyrimidine oligodeoxyribonucleotides (ODNs) did not show appreciable binding. Phosphorothioate modification of purine TFOs did not significantly reduce binding affinity. We also demonstrate that preformed triplexes are quite stable when precipitated with ethanol and resuspended in water. Further analysis was carried out using two purine phosphorothioate antiparallel TFOs, 158 APS and 164 APS, designed to bind to the promoter region from -141 to -158 and -147 to -164, respectively, which were found to form triplexes even under physiological conditions. DNase I footprinting experiments showed the ability of these TFOs to protect target sequences in the promoter region; both purine sequences (C1 and C2) were protected in the case of 158 APS. Transfection experiments using preformed triplexes with a reporter plasmid containing the collagen promoter sequence showed significant inhibition of transcription when compared with a control phosphorothioate ODN. The effect of 164 APS was greater than that of 158 APS. These results indicate that this triplex strategy could be used in the down-regulation of collagen synthesis in cultured cells and offer the potential to control fibrosis in vivo.

Animals↗
PubMed1997

2',5'-linked oligo-3'-deoxyribonucleoside phosphorothioate chimeras: thermal stability and antisense inhibition of gene expression.

2',5'-Linked oligo-3'-deoxyribonucleotides bind selectively to complementary RNA but not to DNA. These oligonucleotides (ODNs) do not recognize double-stranded DNA by Hoogsteen triplex formation and the complexes formed by these ODNs with RNA are not substrates for Escherichia coli RNase H. Substitution of the 2',5'-phosphodiester backbone by phosphorothioate linkages gives 2',5'-linked oligo-3'-deoxynucleoside phosphorothioate ODNs that exhibit significantly less non-specific binding to cellular proteins or thrombin. Incorporation of a stretch of seven contiguous 3',5'-linked oligo-2'-deoxynucleoside phosphorothioate linkages in the center of 2',5'-linked ODNs (as a putative RNase H recognition site) afford chimeric antisense ODNs that retain the ability to inhibit steroid 5alpha-reductase (5alphaR) expression in cell culture.

Base Composition↗
PubMed2000

Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation.

Several studies have shown improved efficacy of cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotides. To gain insight into the mechanisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3'-cholesterol analog of the ICAM-1-specific phosphorothioate oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [(3)H]ISIS-9388 was cleared from the circulation with a half-life of 49.9 +/- 2.2 min (ISIS-3082, 23.3 +/- 3.8 min). At 3 h after injection, the liver contained 63.7 +/- 3. 3% of the dose. Compared to ISIS-3082, the hepatic uptake of ISIS-9388 is approximately 2-fold higher. Endothelial, Kupffer and parenchymal cells accounted for 45.7 +/- 5.7, 33.0 +/- 5.9 and 21.3 +/- 2.6% of the liver uptake of [(3)H]ISIS-9388, respectively, and intracellular concentrations of approximately 2, 75 and 50 microM, respectively, could be reached in these cells (1 mg/kg dose). Preinjection with polyinosinic acid or poly-adenylic acid reduced the hepatic uptake of [(3)H]ISIS-9388, which suggests the involvement of (multiple) scavenger receptors. Size exclusion chromatography of mixtures of the oligonucleotides and rat plasma indicated that ISIS-9388 binds to a larger extent to high molecular weight proteins than ISIS-3082. Analysis by agarose gel electrophoresis indicated that ISIS-9388 binds more tightly to plasma proteins than ISIS-3082. The different interaction of the oligonucleotides with plasma proteins possibly explains their different dispositions. We conclude that cholesterol conjugation results in high accumulation of phosphorothioate oligodeoxynucleotides in various liver cell types, which is likely to be beneficial for antisense therapy of liver-associated diseases.

Animals↗
PubMed2003

Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA.

Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA-DNA-LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2'-O-methyl RNA-DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA-DNA-LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1-green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC50 of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC50 of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC50 approximately 70 nM). In contrast, the efficiency of a 2'-O-methyl-modified oligonucleotide (IC50 approximately 220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA-DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.

Animals↗
PubMed1979

Crystal and molecular structure of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester (Sp diastereomer). Substrate stereospecificity of snake venom phosphodiesterase.

Phosphorylation of 2',3'-O-methoxymethylidene adenosine with bis(p-nitrophenyl) O,O-phosphorochloridothioate, followed by alkaline and acidic deprotection, afforded a mixture of the Rp and Sp diastereomers of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester in good yield. Only one of these diastereomers is a substrate for snake venom phosphodiesterase. The other diastereomer, remaining after complete enzymatic digestion of the substrate, was crystallized as the triethylammonium salt in the triclinic space group P1. The structure was solved from three-dimensional X-ray data and refined to an R value of 5.2%. The asymmetric cell unit contains two independent molecules with almost identical conformations. The ribose is puckered C(2')-endo, the heterocycle is in anti position and the C(5')-O(5') bond gauche, gauche. The triethylammonium cation is coordinated to the free oxygen of the phosphorothioate group and the P-S bond has double bond character. The absolute configuration of the phosphorous is Sp. This is in agreement with an earlier stereochemical study of the hydrolysis of the diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate by snake venom phosphodiesterase.

Adenine Nucleotides↗