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Results for “Phosphorothioate Oligonucleotides”

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At least 19 recordsLinked to original sources

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↗

PubMed2005

Characterization of high molecular weight impurities in synthetic phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides manufactured by standard phosphoramidite techniques using 2'-deoxyadenosine- or 2'-O-(2-methoxyethyl)-5-methylcytosine-loaded solid supports contain branched impurities consisting of two chains linked through the exocyclic amino group of the 3'-terminal nucleoside of one chain and the 3'-terminal hydroxyl group of another via a P(O)SH group. These impurities are not produced when a universal, non-nucleoside derivatized support is used.

5-Methylcytosine↗

PubMed2000

The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides.

Phosphorothioate oligodeoxynucleotides (sODN) containing the CpG motif or TCG repeats induce T cell-independent polyclonal activation of human B cells. To elucidate the mechanism of this response, the role of cell surface receptors was investigated. Sepharose beads coated with stimulatory but not nonstimulatory sODNs induced B cell proliferation comparably with soluble sODNs. The B cell stimulatory activity of Sepharose-bound sODN did not result from free sODN released from the beads since media incubated with coated beads were inactive. Using FITC-labeled sODNs as probes, binding to human B cells could be detected by flow cytometry. Binding was rapid, saturable, initially temperature independent, but with a rapid off-rate. Competition studies indicated that both stimulatory sODNs and minimally stimulatory sODNs bound to the same receptor. By contrast, phosphodiester oligonucleotides with the same nucleotide sequence as sODNs and bacterial DNA inhibited the binding of sODNs to B cells minimally. Charge appeared to contribute to the binding of sODNs to B cells since binding of sODNs was competitively inhibited by negatively charged molecules, including fucoidan, poly I, and polyvinyl sulfate. These data indicate that human B cells bind sODNs by a receptor-mediated mechanism that is necessary but not sufficient for polyclonal activation.

Adult↗

PubMed1993

Inhibition of restriction endonuclease cleavage site via triple helix formation by homopyrimidine phosphorothioate oligonucleotides.

The ability of pyrimidine rich oligonucleotide phosphorothioate to form stable triple helical structures with the sequence containing the recognition site for the class II-S restriction enzyme Ksp 632-I was examined. First, we synthesized double strand oligonucleotides corresponding to the SV40 sites and studied their interaction with homopyrimidine oligodeoxyribonucleotides including replacement of the other chain either PS group (SO-ODNs) in second nucleotide position (from 5'-terminus) and end capped with the PS group at both 3'- and 5'-ends (S2O-ODNs). The resulting perfect DNA triplexes were detected by gel-mobility shift. The phosphorothioate oligonucleotide analogues (SO-ODNs) and (S2O-ODNs) were shown to inhibit enzymatic cleavage under conditions that allow for triple helix formation. Inhibition is sequence-specific and occurs in the micromolar concentration range. Of particular interest is the Sp-phosphorothioate analogue (Sp-SO-ODNs) which inhibited endonuclease more than the other phosphorothioate oligonucleotide analogues (Rp-SO-ODNs or S2O-ODNs).

Base Sequence↗

PubMed1995

[Reactive derivatives of oligonucleotide phosphorothioate analogs. I. Selective activation of terminal phosphates and preparation of phosphamide derivatives of oligonucleotide phosphorothioate analogs].

Selective activation of the terminal phosphates of oligonucleotide analogs containing internucleotide phosphorothioate groups was carried out using the triphenylphosphine-dipyridyldisulfide reducing-oxidizing pair. This formed the basis for a new method of synthesizing various derivatives of phosphorothioate oligonucleotide analogs. Using this method, zwitterionic 3'-phosphamide derivatives of di- and trinucleotide phosphorothioates TpsTp and TpsTpsTp containing the 4-dimethylaminopyridine or N-methylimidazole residues were synthesized. The derivatives reacted almost quantitatively with amines to form, within 5-10 min, the corresponding 3'-phosphamides without cleavage of the internucleotide phosphorothioate groups. Alkylating derivatives of phosphorothioates TpsTp(N(CH3)CH2RCl) and TpsTpsTp(N(CH3)CH2RCl) (RCl = -C6H4N(CH3)CH2CH2Cl) were also synthesized by the proposed method. The internucleotide phosphorothioate residues of the oligonucleotide analogs were stable during the synthesis in DMF. The activity of the alkylating RCl groups in the synthesized compounds was maintained at 85-90%.

Dimethoate↗

PubMed1997

The multiple inhibitory mechanisms of GEM 91, a gag antisense phosphorothioate oligonucleotide, for human immunodeficiency virus type 1.

GEM 91 (gene expression modulator) is a 25-mer oligonucleotide phosphorothioate complementary to the gag initiation site of HIV-1. GEM 91 has been studied in various in vitro cell culture models to examine inhibitory effects on different stages of HIV-1 replication. Experiments were focused on the binding of virions to the cell surface, inhibition of virus entry, reverse transcription (HIV DNA production), inhibition of steady state viral mRNA levels, inhibition of virus production from chronically infected cells, and inhibition of HIV genome packaging within virions. Experiments were also performed in vitro in an attempt to generate strains of HIV with reduced sensitivity to GEM 91. We observed sequence-dependent inhibition of virus entry/reverse transcription and a reduction in steady state viral RNA levels. We also observed sequence-independent inhibition of virion binding to cells and inhibition of virus production by chronically infected cells. Using in vitro methods that were successful in generating HIV strains with reduced sensitivity to AZT, we were unable to generate strains with reduced sensitivity to GEM 91.

Anti-HIV Agents↗

PubMed1991

In vivo studies with phosphorothioate oligonucleotides: pharmacokinetics prologue.

Phosphorothioate oligonucleotides which contain 35S at each internucleoside linkage have been prepared and employed to evaluate the in vivo pharmacokinetics in mice, rats and rabbits. A single administration of a 27-mer complementary to the rev gene of HIV into adult male rats by either the intravenous or intraperitoneal route reveals a biphasic plasma elimination. An initial half-life of 15-25 min represents distribution out of the plasma compartment and a second half-life of 20-40 h represents elimination from the body. The second half-life is significantly longer than a variety of nucleic acids such as poly-IC and Ampligen and suggests therapy with phosphorothioate oligonucleotides should be possible and practical. Repeated daily injections of the 27-mer provides steady-state concentrations in 6-9 days, confirming the estimated long half-life from single injection studies. Finally, chronic treatment studies indicate that the phosphorothioate oligonucleotides are relatively non-toxic. Hence, pharmacokinetic considerations are not likely to be limiting factors in anti-cancer drug design with phosphorothioate oligonucleotides.

Animals↗

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↗

PubMed1990

Optimization of the efficiency of cross-linking PtII oligonucleotide phosphorothioate complexes to complementary oligonucleotides.

We have investigated the efficiency with which PtII complexes cross-link phosphorothioates of oligonucleotides to complementary DNA targets. The A and G residues 2-5 bases downstream from the 5'-phosphorothioate group are preferred sites for cross-linking. Replacement of residues in this part of the target by T residues results in greatly decreased cross-linking when cis platinum diammine dichloride (cisPtII) or potassium platinous chloride (K2PtCl4) are used. Trans platinum diammine dichloride (transPtII) forms cross-links with T residues if A and G residues are absent from the susceptible region of the target. Oligomers containing an internal phosphorothioate group can also be linked to their templates with transPtII, but not with cisPtII or K2PtCl4. Cross-linking via an internal phosphorothioate group tends to be less efficient than cross-linking via a 5'-terminal phosphorothioate. The Sp isomers of internal phosphorothioates are cross-linked more efficiently than the Rp isomers. Preliminary experiments suggest that the efficiency of cross-linking to RNA targets will prove similar to that found for DNA targets.

Base Sequence↗

PubMed1997

[Reactive derivatives of phosphorothioate oligonucleotide analogues. II. Properties of phosphorothioate oligodeoxyribonucleotide derivatives containing an aromatic residue of nitrogen mustard gas].

The alkylating properties of the derivatives of phosphorothioate oligonucleotide analogues containing the alkylating 3'-[N-methyl-4-(N'-methyl-N'-2-chloroethylamino)benzyl]phosphamide group were studied using the alkylating 3'-phosphamide derivative of trithymidine phosphorothioate TpsTpsTpN(CH3)CH2RCl. It was shown by ion-exchange chromatography and 31P NMR spectroscopy that reagents of this type are able to alkylate, depending on the conditions, both their own internucleotide phosphorothioate residues and nucleophiles in the medium. In the absence of external nucleophiles in aqueous media, TpsTpsTpN(CH3)CH2RCl forms predominantly the products of intramolecular alkylation of ps-groups rather than the products of hydrolysis of the RCl-group. In the presence of a nucleophile (1 M aqueous ethylenediamine), the reagent alkylates mainly the aliphatic amino groups of the nucleophile. If the ethylenediamine concentration is reduced to 0.1 M, both processes take place, whereby the rate of reaction with EDA is twice as high as that of intramolecular alkylation. The rate constant of the limiting stage of alkylation (ionization of the C-Cl bond), k0, does not depend on the presence of the competitive nucleophile (ethylenediamine) and is 3.95 x 10(-4) s-1 at 37 degrees C.

Alkylating Agents↗

PubMed1997

Specific inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by liposomally endocapsulated antisense phosphorothioate oligonucleotides: penetration and localization of oligonucleotides in clone 76 cells.

Liposomally encapsulated phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The liposomally encapsulated phosphorothioate oligonucleotides (S-ODNs) complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. Displacement of the target AUG initiation codon sequence to the 3'-end, 5'-end, and/or center sites on the antisense phosphorothioate oligonucleotides was studied with regard to the inhibition of influenza virus RNA polymerases and NP. The antisense phosphorothioate oligonucleotide containing the AUG initiation codon at the center site of the oligonucleotide had the highest inhibitory effects. The liposomally encapsulated phosphorothioate oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. Observation of clone 76 cells treated with the endocapsulated antisense phosphodiester oligonucleotide, FITC-ODNs-PB2-T3, by a confocal laser scanning microscope, revealed diffuse fluorescence, apparently within the cytoplasm. Interestingly, the endocapsulated antisense phosphorothioate oligonucleotide, FITC-S-ODNs-PB2-T3 accumulated in the nuclear region of clone 76 cells. However, weak fluorescence was observed in the endosomes and in the cytoplasms of the clone 76 cells treated with the free antisense phosphorothioate oligonucleotides.

Animals↗

PubMed1998

Rapid induction of Jak2 and Sp1 in T cells by phosphorothioate oligonucleotides.

Phosphorothioate-modified ODNs ([S]ODNs) are known to exert a variety of sequence-independent effects that are mediated in part by rapid induction of the Sp1 transcription factor. An unidentified tyrosine kinase was implicated in this Sp1 induction. In the present study, antisense [S]ODNs, initially designed to target three signaling molecules in the prolactin (PRL)-responsive rat Nb2 T cell line rapidly elevated Jak2 tyrosine kinase and Sp1 protein levels. The [S]ODN-mediated elevation of Jak2 peaked (3-fold to 6.5-fold above controls) at 15 minutes and returned to basal levels by 1 hour, whereas elevation of Sp1 (about 2-fold above controls) peaked at 1 hour. The [S]ODN-mediated induction of Sp1, but not Jak2, was abrogated by AG 490, a Jak2-specific inhibitor. In the presence of submaximal doses of PRL (0.18-0.36 ng/ml), [S]ODN-mediated induction of Jak2 and Sp1 was sustained for 72 hours. Furthermore, the [S]ODNs alone significantly increased Nb2 cell growth and enhanced the growth stimulatory effects of PRL on these cells. In contrast, unmodified ODNs had no effect on Jak2 or Sp1 protein levels and did not stimulate Nb2 cell growth. In conclusion, [S]ODNs stimulate the coordinate induction of Jak2 and Sp1 and stimulate Nb2 T cell proliferation in a sequence-independent manner. The abrogation of Sp1 induction by AG 490 indicates that Jak2 tyrosine kinase is required for [S]ODN-mediated induction of Sp1 in these cells. These results may help to explain some of the nonspecific effects of [S]ODNs, particularly in cytokine-dependent immune cells.

Cell Cycle Proteins↗

PubMed1993

In vitro evaluation of phosphorothioate oligonucleotides targeted to the E2 mRNA of papillomavirus: potential treatment for genital warts.

Papillomaviruses induce benign proliferative lesions, such as genital warts, in humans. The E2 gene product is thought to play a major role in the regulation of viral transcription and DNA replication and may represent a rational target for an antisense oligonucleotide drug action. Phosphorothioate oligonucleotides complementary to E2 mRNAs were synthesized and tested in a series of in vitro bovine papillomavirus (BPV) and human papillomavirus (HPV) models for the ability to inhibit E2 transactivation and virus-induced focus formation. The most active BPV-specific compounds were complementary to the mRNA cap region (ISIS 1751), the translation initiation region for the full-length E2 transactivator (ISIS 1753), and the translation initiation region for the E2 transrepressor mRNA (ISIS 1755). ISIS 1751 and ISIS 1753 were found to reduce E2-dependent transactivation and viral focus formation in a sequence-specific and concentration-dependent manner. ISIS 1755 increased E2 transactivation in a dose-dependent manner but had no effect on focus formation. Oligonucleotides with a chain length of 20 residues had optimal activity in the E2 transactivation assay. On the basis of the above observations, ISIS 2105, a 20-residue phosphorothioate oligonucleotide targeted to the translation initiation of both HPV type 6 (HPV-6) and HPV-11 E2 mRNA, was designed and shown to inhibit E2-dependent transactivation by HPV-11 E2 expressed from a surrogate promoter. These observations support the rationale of E2 as a target for antiviral therapy against papillomavirus infections and specifically identify ISIS 2105 as a candidate antisense oligonucleotide for the treatment of genital warts induced by HPV-6 and HPV-11.

Animals↗

PubMed1992

Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells.

A phosphorothioate oligonucleotide that has been employed to inhibit HIV-1 viral expression in chronically infected H9 cells was examined for cellular uptake and subcellular distribution. The relationship between extracellular oligonucleotide concentration and the distribution and accumulation into subcellular organelles is important to the design, potential side effects, and understanding of a therapeutically useful antisense oligonucleotide. These studies employed uptake of both 35S- and fluorescence-labeled phosphorothioate oligonucleotides. Experiments with V79, HeLa, H9, and fresh human peripheral blood monocytes indicate that accumulations of oligonucleotide inside cells exceeds the concentration of oligonucleotide in culture media by over 100 times following 1 h of exposure at 37 degrees C. Uptake is more efficient at low concentrations, suggesting a saturable process. The total oligonucleotide that remains in cells begins to reach a plateau after 45-60 min, indicating either that efflux pathways exist or that uptake is saturable. Subcellular fractionation studies with 35S-labeled phosphorothioate demonstrate the oligonucleotide is sequestered into both the nuclei and the mitochondria of cultured HeLa cells in a time-dependent manner. The subcellular fractionation was examined with fluorescence-labeled phosphorothioate by both confocal and fluorescence microscopy, which confirmed the rate and localization of oligonucleotide into cultured cells. Finally, cellular uptake is not uniform for all cells in a nonsynchronous culture.

Animals↗

PubMed2002

Phosphorothioate oligonucleotides, suramin and heparin inhibit DNA-dependent protein kinase activity.

Phosphorothioate oligonucleotides and suramin bind to heparin binding proteins including DNA polymerases, and inhibit their functions. In the present study, we report inhibition of DNA-dependent protein kinase activity by phosphorothioate oligonucleotides, suramin and heparin. Inhibitory effect of phosphorothioate oligonucleotides on DNA-dependent protein kinase activity was increased with length and reached a plateau at 36-mer. The base composition of phosphorothioate oligonucleotides did not affect the inhibitory effect. The inhibitory effect by phosphorothioate oligodeoxycytidine 36-mer can be about 200-fold greater than that by the phosphodiester oligodeoxycytidine 36-mer. The inhibitory effect was also observed with purified DNA-dependent protein kinase, which suggests direct interaction between DNA-dependent protein kinase and phosphorothioate oligonucleotides. DNA-dependent protein kinase will have different binding positions for double-stranded DNA and phosphorothioate oligodeoxycytidine 36-mer because they were not competitive in DNA-dependent protein kinase activation. Suramin and heparin inhibited DNA-dependent protein kinase activity with IC(50) of 1.7 microM and 0.27 microg ml(-1) respectively. DNA-dependent protein kinase activities and DNA double-stranded breaks repair in cultured cells were significantly suppressed by the treatment with suramin in vivo. Our present observations suggest that suramin may possibly result in sensitisation of cells to ionising radiation by inactivation of DNA-dependent protein kinase and the impairment of double-stranded breaks repair.

Antineoplastic Agents↗

PubMedDate not supplied

Investigation of a facile synthetic method for phosphorothioate dimer synthons in oligonucleotide phosphorothioates synthesis.

A facile synthetic method of a phosphorothioate dimer block was investigated. Dinucleoside phosphite triester intermediates were obtained in one-pot synthesis by the coupling of a protected nucleoside bearing free 5'-OH and a protected nucleoside bearing free 3'-OH in the presence of phosphorous trichloride (PCl3) and 1,2,4-triazole. The intermediates were easily sulfurized to afford the desired phosphorothioate dimer blocks in 33-64% overall yields.

Chromatography, Thin Layer↗

PubMed1997

A nonradioisotope approach to study the in vivo metabolism of phosphorothioate oligonucleotides.

A 25-mer phosphorothioate oligodeoxynucleotide (GEM 91) complementary to the gag gene mRNA of HIV-1 virus was administered intravenously (i.v.) at a dose of 10 mg/kg/day for 8 weeks or 25 mg/kg single dose subcutaneously (SC) to adult Rhesus monkeys. No radioactive markers were used. A capillary gel electrophoresis (CGE) method with UV detection was used to determine the concentration of GEM 91 in plasma and the metabolite profile. The metabolite profile was virtually the same following a single dose of either 10 mg/kg i.v. or 25 mg/kg SC. A different metabolite profile was observed after 4 or 8 weeks of multiple i.v. doses of 10 mg/kg/day. The extract was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) for positive identification. Mass spectrometry confirmed the major metabolic pathway in vivo to be via 3'-end exonuclease activity. The extract was then subjected to a hybridization-assisted ligation reaction in which only 5'-end intact metabolites were labeled. Analysis by CGE with laser-induced fluorescence (LIF) detection allowed each of these metabolites to be quantified with a limit of detection of 1 ppb (ng/ml). MALDI-TOFMS identified components digested from both ends of the DNA. This study demonstrates that the combination of quantitative CGE-LIF and MALDI-TOFMS yields a powerful and unique approach to study the metabolism of phosphorothioate oligonucleotides.

Animals↗

PubMed1999

Enzymatic assignment of diastereomeric purity of stereodefined phosphorothioate oligonucleotides.

Enzymatic hydrolysis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-oligos) synthesized via the oxathiaphospholane method has been used for assignment of their diastereomeric purity. For this purpose, two well-known enzymes of established diastereoselectivity, nuclease P1 and snake venom phosphodiesterase (svPDE) have been used. However, because of some disadvantageous properties of svPDE, a search for other [Rp]-specific endonucleases was undertaken. Extracellular bacterial endonuclease isolated from Serratia marcescens accepts PS-oligos as substrates and hydrolyzes phosphorothioate bonds of the [Rp] configuration, whereas internucleotide [Sp]-phosphorothioates are resistant to its action. Cleavage experiments carried out with the use of unmodified and phosphorothioate oligonucleotides of different sequences demonstrate that the Serratia nuclease is more selective in recognition and hydrolysis of oligodeoxyribonucleotides than previously reported. The substrate specificity exhibited by the enzyme is influenced not only by the nucleotide sequence at the cleavage site but also by the length and base sequence of flanking sequences. The Serratia nuclease can be useful for analysis of diastereomeric purity of stereodefined phosphorothioate oligonucleotides, but because of its sequence preferences, the use of this enzyme in conjunction with svPDE is more reliable.

Endodeoxyribonucleases↗