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PubMed2000

Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them?

The development of nucleoside phosphorothioates is described in its historical context. Examples of the interaction of phosphorothioate groups, present either in oligodeoxynucleotides or in DNA, with nucleases are presented. The structural features responsible for the resistance of the phosphorothioates toward degradation by nucleases are discussed, as are the possible reasons for the high-affinity interaction of phosphorothioate oligodeoxynucleotides with certain proteins.

Animals↗
PubMed1985

The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA.

The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.

Base Sequence↗
PubMed1988

Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides.

The Rp- and Sp-diastereomers of the phosphorothioate-containing oligonucleotide d[ApAp(S)ApA] have been synthesized. They and the tetramer d[ApApApA] were tested as substrates for staphylococcal nuclease, DNase II and spleen phosphodiesterase. For digestions with DNase I these oligonucleotides were converted to the 5'-phosphorylated derivates. The reactions with the nucleases were analysed by HPLC. The phosphorothioate groups of both diastereomers were resistant to the action of staphylococcal nuclease, DNase I and DNase II. While the phosphorothioate group of the Rp-diastereomer was resistant to the action of spleen phosphodiesterase, the Sp-diastereomer was hydrolysed at an estimated rate 1/100 the rate of cleavage of the unmodified tetramer. The presence of the phosphorothioate group in the center of the molecule affected the rate of hydrolysis of neighbouring phosphate groups for some enzymes. In particular, very slow release of 3'-dAMP from the Rp-diastereomer occurred on incubation with staphylococcal nuclease but the Sp-diastereomer was completely resistant. DNase II produced 3'-dAMP quite rapidly from both diastereomers of d[ApAp(S)ApA] and DNase I released 5'-dAMP from both diastereomers of d[pApAp(S)ApA] only slowly.

Animals↗
PubMed1989

Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution.

The group I intron from the rRNA precursor of Tetrahymena undergoes self-splicing. The intron RNA catalyst contains about 400 phosphate groups. Their role in catalysis has been investigated using phosphorothioate substituted RNA. In such RNA one of the peripheral oxygens of the phosphodiesters is replaced with sulfur. Incorporation of adenosine 5' phosphorothioate in either the 5' or 3' half of the ribozyme blocked splicing whereas incorporation of uridine 5' phosphorothioate only blocked splicing if the substitution was in the 3' half of the molecule. Modification-interference assays located two major and three minor inhibitory phosphorothioate substitutions suggesting that the corresponding phosphates play a significant role in self-splicing. These are all located in the most highly conserved region of the intron.

Adenosine Monophosphate↗
PubMed1990

The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos.

The properties of antisense phosphorothioate and unmodified oligodeoxynucleotides have been studied in Xenopus oocytes and embryos. We find that phosphorothioates, like unmodified oligodeoxynucleotides, can degrade Vg1 mRNA in oocytes via an endogenous RNase H-like activity. In oocytes, phosphorothioate oligodeoxynucleotides are more stable than unmodified oligodeoxynucleotides and are more effective in degrading Vg1 mRNA. In embryos, neither unmodified nor phosphorothioate deoxyoligonucleotides were effective in degrading Vg1 message at sub-toxic doses.

Animals↗
PubMed1996

Solid support synthesis of all-Rp-oligo(ribonucleoside phosphorothioate)s.

The first method for solid support synthesis of all-Rp-oligo(ribonucleoside phosphorothioate)s is presented as well as attempts to increase the stereoselectivity of the key step in this approach. The synthetic strategy consists of (i) a solid support synthesis procedure, using 5'-O-(4-methoxytriphenylmethyl)-2'-O-tert-butyldimethylsilyl-ri bon ucleoside 3'-H- phosphonates, that due to stereoselectivity in the condensation step, gives oligomers with mostly Sp-H-phosphonate diesters (72-89% under standard conditions), (ii) stereospecific sulfurization with S8 in pyridine to produce oligo(ribonucleoside phosphorothioate)s enriched with internucleosidic linkages of Rp configuration, (iii) treatment of the deprotected oligonucleotides with the enzyme Nuclease P1 from Penicillium citrinum, that specifically catalyses cleavage of Sp-phosphorothioate diester linkages, which leaves a mixture of oligomers having all internucleosidic linkages as Rp-phosphorothioates, and finally (iv) isolation and HPLC purification of the full length all-Rp oligomer. Mixed sequences containing the four common nucleosidic residues up to the chain length of a heptamer were synthesized. Change of N-4-protection on the cytidine building block from propionyl to N-methylpyrrolidin-2-ylidene gave a slightly improved diastereoselectivity in H-phosphonate diester formation. Increased selectivity up to 99+% was obtained with the guanosine building block when the amount of pyridine in the coupling step was reduced.

Chromatography, High Pressure Liquid↗
PubMed1996

NMR investigations of duplex stability of phosphorothioate and phosphorodithioate DNA analogues modified in both strands.

Duplex formation from the self-complementary 12mer d(CGCGAATTCGCG) (Dickerson dodecamer) in which all phosphodiester linkages were replaced by phosphorothioate or phosphorodithioate linkages was studied using variable-temperature 1H and 31P NMR spectroscopy. Melting temperatures of the dodecamer, measured spectrophotometrically, showed significant decrease upon sulfur substitution (Tm 49 degrees C for the phosphorothioate and 21 degrees C for the phosphorodithioate, compared with 68 degrees C for the unmodified oligomer, in 1 M salt). Hyperchromicity observed upon melting of the dithioate was surprisingly low. NOESY spectra of the monothioate showed a cross-peak pattern characteristic for a right-handed duplex. Imino proton resonances of the duplex, shown by the mono- and the dithioate, were similar to those of the parent compound. In spite of monophasic melting curves, temperature dependence of the imino proton resonances and phosphorus resonances of the phosphorodithioate indicated heterogeneity with respect to base-pairing, compatible with the presence of a hairpin loop. Relaxation times (T1) of the imino protons in the phosphorothioate, determined by the saturation recovery method, were considerably shorter than in the unmodified oligomer. Base-pair lifetimes in the unmodified Dickerson dodecamer, determined by catalyst-dependent changes in relaxation rates of imino protons, were in the range of 2-30 ms at 20 degrees C. Strongly reduced base-pair lifetimes were found in the phosphorothioate analogue.

DNA↗
PubMed1997

In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.

Animals↗
PubMed1997

CD, absorption and thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes [d/r(AC)]12.[d/r(GT/U)]12 and the influence of phosphorothioate substitution.

Circular dichroism (CD) spectra and melting temperature (Tm) data for five duplexes containing phosphorothioate linkages were compared with data for four unmodified duplexes to assess the effect of phosphorothioate modification on the structure and stability of DNA. DNA and DNA.RNA duplexes. Nine duplexes were formed by mixing oligomers 24 nt long in 0.15 M K+(phosphate buffer), pH 7.0. Unmodified DNA.DNA and RNA.RNA duplexes were used as reference B-form and A-form structures. The CD spectra of the modified hybrids S-d(AC)12.r(GU)12 and r(AC)12.S-d(GT)12 differed from each other but were essentially the same as the spectra of the respective unmodified hybrids. They were more A-form than B-form in character. CD spectra of duplexes S-d(AC)12.d(GT)12 and d(AC)12.S-d(GT)12 were similar to that of d(AC)12.d(GT)12, except for a reduced long wavelength CD band. Sulfur modifications on both strands of the DNA duplex caused a pronounced effect on its CD spectrum. The order of thermal stability was: RNA.RNA > DNA.DNA > DNA.RNA > S-DNA.DNA > S-DNA. RNA > S-DNA.S-DNA. Phosphorothioation of one strand decreased the melting temperature by 7.8+/-0.6 degrees C, regardless of whether the substitution was in a hybrid or DNA duplex. Thermodynamic parameters were obtained from a multistate analysis of the thermal melting profiles. Interestingly, the destabilizing effect of the phosphorothioate substitution appears to arise from a difference in the entropy upon forming the DNA.DNA duplexes, while the destabilizing effect in the DNA.RNA hybrids appears to come from a difference in enthalpy.

Circular Dichroism↗
PubMed1998

Incorporation of terminal phosphorothioates into oligonucleotides.

Considerable effort has been directed towards studying the structure and function of oligonucleotides and several approaches rely on the attachment of reporter groups to oligonucleotides. We report here the introduction of 3'- and 5'-terminal phosphorothioates into heptameric oligonucleotides and their post-synthetic modification with several reporter groups. The synthesis of terminal phosphorothioates is based on the coupling of a ribonucleoside phosphoramidite at the first or last nucleotide, respectively, which, after sulphurization, is removed by sequential oxidation of the vicinal hydroxyl groups and then beta-elimination. Product formation is of the order of 95%. The ratio of phosphorothioate- versus phosphate-terminated oligodeoxynucleotides as analysed by electrophoresis on a Hg2+gel is in general 85/15. Examples for the reactivity of the terminal phosphorothioates for conjugation with cholesterol, bimane and for sulphydryl exchange are described.

Bridged Bicyclo Compounds↗
PubMed2000

Influence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: a circular dichroism study.

Phosphorothioate oligodeoxynucleotides (ODNs) have been extensively investigated in vivo and in vitro for antisense control of gene expression. It has been shown that cellular uptake of phosphorothioate ODNs in some in vitro cell systems increases in the presence of divalent cations. In this work, we analyze the conformation of phosphorothioate ODNs and specific changes induced in it by various divalent cations using circular dichroism (CD) spectroscopy. CD data were obtained with several phosphorothioate ODNs in the absence and presence of the divalent cations Mg(2+), Ca(2+), Sr(2+), Ba(2+) and Mn(2+). All CD spectra indicated stable conformations of the ODNs in solution. The spectra were strongly dependent on ODN sequence and composition. Some ODNs such as T(23) and another with 'random' distribution of bases showed CD spectra characteristic of B-form DNA. Other ODNs which had at least three consecutive guanines in their sequences exhibited spectra characteristic of parallel G-tetraplexes. CD spectra of antisense ODNs exhibited specific responses to divalent cations. Changes in the conformation were not simply due to ionic strength effects. Mn(2+) diminished secondary structure in some ODNs. Group II divalent ions stabilized the parallel G-tetraplexes, and Mg(2+) generally had the weakest stabilizing efficiency. Each sequence/ion combination had a specific response so these effects cannot be generalized. These sequence-dependent, divalent ion-sensitive, and structurally unique solution conformations may be related to ion-mediated ODN uptake.

Barium↗
PubMed2002

The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2'-O-methyl modifications.

One of the problems that hamper the use of antisense DNAs as effective drugs is the non-specific binding of chemically-modified oligonucleotides to cellular proteins. We previously showed that the affinity of a model ssDNA-binding protein, the Ff gene 5 protein (g5p), was >300-fold higher for phosphorothioate-modified DNA (S-DNA) than for unmodified dA(36), consistent with the propensity of S-DNA to bind indiscriminately to proteins. The current work shows that g5p binding is also sensitive to sugar and pyrimidine modifications used in antisense oligomers. Binding affinities of g5p for 10 36mer oligomers were quantitated using solution circular dichroism measurements. The oligomers contained C-5-propyne (prC), 2'-O-methyl (2'-O-Me) or 2'-OH (RNA) groups, alone or combined with the phosphorothioate modification. In agreement with reported increases in antisense activity, the addition of prC or 2'-O-Me modifications substantially reduced the affinity of oligomers for g5p by approximately 2-fold compared with the same DNA oligomer sequences containing only phosphorothioate linkages. That is, such modifications moderated the propensity of the phosphorothioate group to bind tightly to the g5p. The Ff g5p could be a useful model protein for assessing non-specific binding effects of antisense oligomer modifications.

Circular Dichroism↗
PubMed2006

Solid-phase synthesis of stereoregulated oligodeoxyribonucleoside phosphorothioates.

An efficient stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates on a solid-support by an oxazaphospholidine method was achieved. A detailed investigation on the reagents and conditions for the solid-phase synthesis revealed that the use of the capping and sulfurizing reagents which are regularly used for the conventional synthesis of phosphorothioate DNA resulted in a sluggish removal of the chiral auxiliaries on the phosphorothioate internucleotidic linkages. After optimization of the capping and sulfurizing steps, the deprotection of the internucleotidic linkages were successfully conducted under the regular basic conditions, and P-stereoregulated phosphorothioate DNA oligomers were successfully obtained with excellent diastereopurity.

Oligodeoxyribonucleotides↗
PubMed2006

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP.

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K(i) between 0.2 and 0.5 microM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 microM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 microM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.

Adenosine Diphosphate↗
PubMed2000

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo.

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.

Adjuvants, Immunologic↗
PubMedDate not supplied

Transcription of 2'-deoxy-2'-fluoro-modified and phosphorothioate-modified RNA templates by HIV-1 reverse transcriptase.

RNA templates yield the corresponding DNA in the presence of human immunodeficiency virus reverse transcriptase (HIV-1 RT). The purpose of this study was to determine whether RNA that was modified with either 2'-deoxy-2'-fluoro analogs or with internucleotide phosphorothioate linkages could serve as templates for HIV-1 RT. Modified RNA that contained either 2'-deoxy 2'-fluoro pyrimidine nucleoside analogs or internucleotide phosphorothioate diester linkages 5'- to pyrimidine nucleosides were enzymatically synthesized and tested for template activity with recombinant HIV-1 RT. RNA that was modified with either 2'-deoxy-2'-fluorouridine or with internucleotide phosphorothioate linkages 5'- to pyrimidines yielded full length HIV-1 reverse transcription products, with complete fidelity in transcription. RNA that was modified with 2'-deoxy-2'-fluorocytidine, either alone or in combination with 2'-deoxy-2'-fluorouridine, did not function as templates for HIV-1 RT, under the conditions reported here. The ability of 2'-deoxy-2'-fluoro-modified and phosphorothioate-modified RNA to serve as template for the RNA-dependent DNA polymerase of HIV-1 RT has not hitherto been reported.

Base Sequence↗
PubMed1991

Phosphorothioate oligodeoxycytidine interferes with binding of HIV-1 gp120 to CD4.

In addition to their properties as sequence-specific inhibitors of gene expression, sequence nonspecific phosphorothioate oligodeoxynucleotides have been shown to protect against the cytopathic effects of HIV-1. Although these compounds are effective inhibitors of HIV-1 reverse transcriptase in vitro, it is not certain that they exert their cytoprotective effect only in this manner. Initial binding of the HIV-1 virion to cells involves the interaction of the viral envelope protein gp120 with CD4. In this report, we describe flow cytometric data and a solid-phase ELISA assay that document the ability of a phosphorothioate deoxycytidine 28-mer to interfere with this interaction by competing with gp120 binding to CD4. The biological importance of this interaction is demonstrated by the fact that phosphorothioate oligodeoxycytidine inhibits syncytium formation resulting from HIV-1-induced cell fusion. These data suggest that phosphorothioate oligodeoxynucleotides may exert their cytoprotective effects, perhaps at least in part, by interfering with the binding of HIV-1 to the target cells.

Antigens, CD↗
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↗