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PubMed1988

Accurate in vitro cleavage by RNase III of phosphorothioate-substituted RNA processing signals in bacteriophage T7 early mRNA.

To test the ability of an RNA processing enzyme to cleave chemically-modified RNA substrates, RNA transcripts containing RNase III cleavage sites were enzymatically synthesized in vitro to contain specific phosphorothioate diester internucleotide linkages. One transcript (R1.1 RNA) was generated using phage T7 RNA polymerase and a cloned segment of phage T7 DNA containing the R1.1 RNase III processing site. The second transcript was the phage T7 polycistronic early mRNA precursor, which was synthesized using E. coli RNA polymerase and T7 genomic DNA. The RNA transcripts contained phosphorothioate diester groups at positions including the scissile bonds. The modified RNAs were stable to incubation in Mg2+-containing buffer, and were specifically cleaved by RNase III. RNA oligonucleotide sequence analysis showed that the modified R1.1 RNA processing site was the same as the canonical site and contained a phosphorothioate bond. Furthermore, RNase III cleaved the phosphorothioate internucleotide bond with 5' polarity. RNase III cleavage of phosphorothioate substituted T7 polycistronic early mRNA precursor produced the same gel electrophoretic pattern as that obtained with the control transcript. Thus, RNase III cleavage specificity is not altered by phosphorothioate internucleotide linkages.

Base Sequence↗
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↗
PubMed1995

Stereodifferentiation--the effect of P chirality of oligo(nucleoside phosphorothioates) on the activity of bacterial RNase H.

P stereoregular phosphorothioate analogs of pentadecamer 5'-d(AGATGTTTGAGCTCT)-3' were synthesized by the oxathiaphospholane method. Their diastereomeric purity was assigned by means of enzymatic degradation with nuclease P1 and, independently, with snake venom phosphodiesterase. DNA-RNA hybrids formed by phosphorothioate oligonucleotides (PS-oligos) with the corresponding complementary pentadecaribonucleotide were treated with bacterial RNase H. The DNA-RNA complex containing the PS-oligo of [all-RP] configuration was found to be more susceptible to RNase H-dependent degradation of the pentadecaribonucleotide compared with hybrids containing either the [all-SP] counterpart or the so called 'random mixture of diastereomers' of the pentadeca(nucleoside phosphorothioate). This stereodependence of RNase H action was also observed for a polyribonucleotide (475 nt) hybridized with these phosphorothioate oligonucleotides. The results of melting studies of PS-oligo-RNA hybrids allowed a rationalization of the observed stereodifferentiation in terms of the higher stability of heterodimers formed between oligoribonucleotides and [all-RP]-oligo(nucleoside phosphorothioates), compared with the less stable heterodimers formed with [all-SP]-oligo(nucleoside phosphorothioates) or the random mixture of diastereomers.

Bacteria↗
PubMed1996

Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynucleotides.

Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A) subunit of the NF-kappaB nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhesion. We studied the effects of Rel A antisense, Rel A sense and other phosphorothioate oligodeoxynucleotides on cellular adhesion and found that blockade of adhesion was predominately non-sequence specific. Phosphorothioate oligodeoxynucleotides bind to the extracellular matrix (ECM) of NIH 3T3 cells, and to the ECM elements laminin and fibronectin. By use of a gel mobility shift assay, the association of the A subunit of laminin with a probe 12mer phosphodiester oligodeoxynucleotide could be demonstrated. This interaction was described by a single-site binding equation (K d = 14 microM). Human Rel A antisense and sense oligodeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleotide binding occurred at or near the heparin-binding site. Competition for 5' 32p- SdT18 (an 18mer phosphorothioate homopolymer of thymidine) binding to fibronectin with the discrete heparin analogs, as well as with SdC28, was also observed. Phosphorothioate oligodeoxynucleotides (Rel A antisense >> Rel A sense) inhibited the binding of laminin to bovine brain sulfatide, but not to its cell surface receptors on MCF-7 cells. By flow cytometric analysis we have also shown, in contrast to what was observed with laminin, that phosphorothioates a non-specifically block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate treated Jurkat cells. Blockade of specific binding occurred in the oligodeoxynucleotide treated cells in the presence or absence of oligomer in the media.

3T3 Cells↗
PubMed1994

The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.

The effect of phosphorothioate bonds on the hydrolytic activity of the 5'-->3' double-strand-specific T7 gene 6 exonuclease was studied. Double-stranded DNA substrates containing one phosphorothioate residue at the 5' end were found to be hydrolyzed by this enzyme as efficiently as unmodified ones. The enzyme activity was, however, completely inhibited by the presence of four phosphorothioates. On the basis of these results, a method for the conversion of double-stranded PCR products into full-length, single-stranded DNA fragments was developed. In this method, one of the PCR primers contains four phosphorothioates at its 5' end, and the opposite strand primer is unmodified. Following the amplification, the double-stranded product is treated with T7 gene 6 exonuclease. The phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed. When the phosphorothioated PCR primer is 5' biotinylated, the single-stranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate. We also describe a simple and efficient method for the immobilization of relatively short oligonucleotides to microtiter plates with a hydrophilic surface in the presence of salt.

Base Sequence↗
PubMed1985

Substrate specificity and stereospecificity of calf spleen phosphodiesterase towards deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates.

Phosphodiesterase from calf spleen exhibits nucleotidyltransferase activity when incubated with either the (PR) or the (PS) diastereomer of thymidyl 3'-(4-nitrophenyl phosphorothioate). Thymidylyl(3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate) was identified as the main product of the enzyme-catalyzed reaction and the absolute configuration at the internucleotide phosphorus atom of the product was determined. The nucleotidyltransferase reaction is shown to proceed with retention of configuration at phosphorus, implying involvement of a double displacement mechanism with the formation of a nucleotidylated enzyme intermediate. To study the substrate specificity of spleen phosphodiesterase a series of deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates were synthesized, and Km and V parameters for each substrate were measured. The results obtained show virtually no specificity for substrates with different nucleosidyl moieties, while about a 20 - 30-fold drop in V and a slight increase in Km values is observed for phosphorothioate analogues as compared with corresponding phosphates. The enzyme showed no significant stereoselectivity towards phosphorothioates of opposite configurations at phosphorus.

Animals↗
PubMedDate not supplied

O,O,S-trimethyl phosphorothioate increases Ca2+ independent nitric oxide synthase activity in the lung but decreases Ca2+/calmodulin dependent type in the cerebellum in Fischer 344 rats.

In the present study, we investigated the possible role of nitric oxide synthase in lung injury using female Fischer 344 rats as a model animal and O,O,S-trimethyl phosphorothioate as an example of lung toxicants. One form of nitric oxide synthase, Ca2+/calmodulin dependent type, decreased monotonously in a dose-dependent manner in the cerebellum. In contrast, O,O,S-trimethyl phosphorothioate increased activities of Ca2+ independent nitric oxide synthase in the lung in a dose-associated manner from 5 mg/kg to 15 mg/kg, but decreased at 30 mg/kg. Lung toxicity of O,O,S-trimethyl phosphorothioate, however, as judged both by functional impairments (PaCO2 and [HCO3-]) and histopathological changes, increased sharply at 30 mg/kg. We thus tested the hypothesis that a potent nitric oxide synthase inhibitor, NG-nitro-L-arginine-methyl ester, may modify lung injury induced by O,O,S-trimethyl phosphorothioate. Treatment with NG-nitro-L-arginine-methyl ester at 20 mg/kg/day aggravated lung injury induced by O,O,S-trimethyl phosphorothioate: Pulmonary oedema and bleeding occurred, leading to an increase in mortalities at 15 mg/kg of O,O,S-trimethyl phosphorothioate, at which level it did not induce such changes as when dosed alone. These findings indicate that nitric oxide synthase in the lung might play a protective role in lung injury.

Amino Acid Oxidoreductases↗
PubMed1985

Bond order and charge localization in nucleoside phosphorothioates.

In the recent literature on nucleoside phosphorothioate anions the structural formulas show a double bond between phosphorus and sulfur and a single bond between phosphorus and oxygen with a negative charge localized on oxygen. However, a review of physical data on these compounds shows the reverse to be the case; that is, in phosphorothioate anions the P-S bond is a single bond with a negative charge localized on sulfur, while the P-O bond order for exocyclic and nonbridging oxygens is greater than 1, approaching 2 in O-alkyl phosphorothioate monoanions and O,O-dialkyl phosphorothioates. The P-O bond orders in phosphorothioate dianions and trianions approach 1 1/2 and 1 1/3, respectively, owing to delocalization of negative charge among two or three oxygens. These conclusions are based on bond lengths obtained from x-ray crystallographic data and electron diffraction, the magnitudes of the effects of 18O on the 31P-nuclear magnetic resonance chemical shifts of phosphorus in nucleoside [18O]phosphorothioates, the pH-dependence of 17O-NMR chemical shifts in [17O]phosphate and [17O]thiophosphate, the vibrational spectra of thiophosphate di- and trianions, and the pKa (dissociation constant) values for phosphoric and thiophosphoric acids.

Chemical Phenomena↗
PubMed1992

Towards rational design of antisense DNA: molecular modelling of phosphorothioate DNA analogues.

In order to assess the effects of substitution of one of the non-bridging oxygens in the phosphodiester group with sulfur, molecular dynamics calculations were performed on duplex oligodeoxynucleotides having a complete helical turn, [d(CpGpCpGpApApTpTpCpGpCpG)]2. The calculations were carried out with the unmodified duplex, as well as with duplexes having one phosphorothioate strand with uniform phosphorus stereochemistry (SP or RP). The electrostatic and structural (equilibrium molecular dimensions as well as force constants) parameters of the phosphorothioate group for molecular mechanics and dynamics calculations were derived from ab initio quantum mechanics optimizations of conformers of O,O'-dimethyl phosphorothioate. Molecular dynamics simulations (30 ps) were carried out using the AMBER force field, with explicit inclusion of all hydrogen atoms, counterions, and water. The simulations indicate a significant decrease of stability of the model containing the homochiral RP-phosphorothioate strand, whereas the model containing the homochiral SP-phosphorothioate strand has practically the same energy as the unmodified oligodeoxynucleotide duplex. The energy difference determined indicates some preference for the use of stereochemically pure SP-phosphorothioates as antisense gene inhibitors. Moreover, the significance of these results in connection with use of phosphorodithioates is discussed.

DNA, Antisense↗
PubMed1979

Chiral [18O]phosphorothioates. The stereochemical course of thiophosphoryl group transfer catalyzed by nucleoside phosphotransferase.

Nucleoside phosphotransferase from barley seedlings was used to catalyze the equilibration of adenosine-5'-[18O]phosphorothioate having the S configuration at phosphorus with [adenine-8-14C]adenosine to produce [adenine-8-14C]adenosine-5'-[18O]phosphorothioate and adenosine. The configuration of the chiral phosphorus in adenosine-5'-[18O]phosphorothioate which was used as the donor substrate was then compared with that of the [adenine-8-14C]adenosine-5'-[18O]phosphorothioate isolated from the reaction mixture. They were found to be the same, showing that the reaction proceeds with 99.7% retention of configuration of the [18O]phosphorothioate. This is interpreted to be indicative of the involvement of a thiophosphoryl-enzyme intermediate in the nucleoside phosphotransferase reaction. The synthesis of adenosine-5'-[18O]phosphorothioate having the R and S configurations at the phosphorus atoms is described.

Adenosine Monophosphate↗
PubMed1994

Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding.

Phosphorothioate modification of internucleoside linkages is widely used to prevent degradation of oligodeoxynucleotide (ODN) therapeutic agents in serum and cells. This modification generally increases ODN potency, but in many instances it is associated with an increase of poorly understood nonspecific effects. In this study, we have found that both cellular retention and nonspecific protein binding are dependent upon the extent of the oligonucleotide's modification. Flow cytometry of cells treated with fluorescein-labeled single-stranded (ss) or double-stranded (ds) ODNs demonstrated that fully phosphorothioate-modified ODNs exhibit much greater cellular association than 3'-terminally modified ODNs (with three 3'-terminal phosphorothioate linkages). Additionally, gel shift assays with either ss- or ds-probes showed that fully phosphorothioate-modified ODNs also exhibit much greater cytoplasmic and nuclear protein binding than either 3'-terminally modified or unmodified ODNs. However, gel shift competition assays showed that transcription factor binding by fully phosphorothioate-modified ds-ODNs was completely nonspecific relative to 3'-terminally modified and unmodified ds-ODNs. These results suggest that the benefits derived from full phosphorothioate modification of ODNs may be negated by increases of nonspecific protein binding and associated sequence-independent effects.

3T3 Cells↗
PubMedDate not supplied

A semi-empirical Hückel molecular orbital analysis of internucleotide phosphorothioate diesters.

Oligonucleotides that contain internucleotide phosphorothioate diesters have potential as anticancer therapeutic agents through sequence-specific antisense mechanisms. Although the chemical reactivity of internucleotide phosphorothioate diesters has been utilized to form phosphorothioate oligonucleotide conjugates, the theoretical basis for this chemical reactivity needs further elucidation. Calculations reported here, performed by the Huckel molecular orbital method, show that the energy of the highest occupied molecular orbital (HOMO) of the internucleotide phosphorothioate diester is 15.9 kcal/mole higher than that of the corresponding HOMO of the internucleotide phosphate diester. The electron density in the phosphorothioate HOMO is almost completely centered on the sulfur atom. The results of this parametricized Huckel molecular orbital analysis are consistent with the model of the internucleotide phosphorothioate sulfur as a soft nucleophile.

Calorimetry↗
PubMed1995

Sequence analysis of phosphorothioate oligonucleotides via matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Modification of the natural phosphodiester backbone of deoxyribooligonucleotides can impart increased biostability via nuclease resistance. Further, uniform incorporation of phosphorothioate linkages renders oligonucleotides highly resistant to reagents traditionally used in sequencing reactions. As a consequence, analytical tests crucial for establishing the identity of such oligonucleotide drugs are less informative. To circumvent this problem, chemical oxidation has been employed for converting the phosphorothioate to the uniform phosphodiester, thereby facilitating enzymatic degradation. Following oxidation, exonucleases which sequentially cleave individual bases from the 3' or 5' terminus of the oligonucleotide or base-specific cleavage chemicals were used to facilitate sequence identification of the oligonucleotide. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), previously used to sequence natural phosphodiester DNA, was then used to sequence the chemically oxidized phosphorothioate. Sequential enzymatic cleavage of desulphurized phosphorothioates in combination with MALDI analysis not only provides a viable alternative to radiolabeling as used in conventional sequencing approaches (e.g. Maxam-Gilbert), but also enables rapid sequencing of phosphorothioate oligonucleotides, for routine drug analysis.

Base Sequence↗
PubMed2000

Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme.

A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G(8) and A(9), a site that is considered to play a crucial role in catalysis, was examined by high-resolution 1H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the (31)P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. (31)P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S-substrate complexes generated in the absence of Mg(2+) ions were still in an inactive form and that Mg(2+) ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification.

Base Sequence↗
PubMed2000

Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes.

The roles of metals in the phosphodiester bond cleavage reaction performed by the hammerhead ribozyme are under investigation. In this study, the apparent affinities and the abilities of several different metals to support ribozyme activity are reported. The relative affinities of divalent cations for the hammerhead ribozyme are determined by measuring their ability to release bound Mn2+. The EPR-detected Mn2+ competition studies give an order of apparent affinity of Mn2+ approximately Co2+ approximately Zn2+ > Cd2+ >> Mg2. This ordering generally follows the trend of maximum rates of cleavage determined at pH 7.0, 0.1 M NaCl, and saturating metal concentrations, of Mn2+ > Co2+ > Cd2+ > Mg2+. The maximum rate is observed for Mn2+ under these conditions and may be related to the high affinity, low pKa and low deltaHhyd of this ion. Substitution of phosphorothioates 5' to each of the nine adenosines in the enzyme strand yields a change in the Mn2+ binding properties of the hammerhead complex. In the phosphorothioate-substituted hammerhead complex, eight to nine Mn2+ bind in two types of classes: 'type 1' (n = 1+/-0.3, Kd = 1.1+/-1 microM) and weaker 'type 2' (n = 7.7+/-0.3, Kd = 125+/-27 microM). The multiple phosphorothioate substitutions result in the loss of two to three of the higher affinity sites observed in the unmodified ribozyme. Metal competition studies with the phosphorothioate-substituted ribozyme indicate that the relative affinities of the metals are Cd2+ > Zn2+ > Co2+, Mg2+ with the number of Mn2+ displaced and apparent affinity of the thiophilic Cd2+ most affected by the phosphorothioate substitutions.

Cadmium↗
PubMed1990

Investigation of the inhibitory role of phosphorothioate internucleotidic linkages on the catalytic activity of the restriction endonuclease EcoRV.

The inhibitory effect of phosphorothioate residues, located within one strand of double-stranded DNA, on the hydrolytic activity of the restriction endonuclease EcoRV was investigated. Specific incorporation of a phosphorothioate group at the site of cleavage yielded the sequence 5'-GATsATC-3'. This modified sequence was cleaved at a relative rate of 0.1 compared to the unmodified substrate. Substrates 5'-GATsAsTC-3' and 5'-GsATsATC-3', both containing one additional phosphorothioate substitution, were linearized at a rate of 0.04 relative to unmodified DNA. However, under the same conditions, fully dAMPS-substituted DNA was found to be virtually resistant to the hydrolytic activity of EcoRV. Further experiments showed that double-stranded DNA fragments generated by PCR containing phosphorothioate groups within both strands are potent inhibitors of EcoRV catalysis. The inhibition was independent of whether the inhibitor fragment contained an EcoRV recognition site. We concluded that substitution of the phosphate group at the site of cleavage by a phosphorothioate residue decreases the rate of EcoRV-catalyzed hydrolysis most significantly. Substitution of other phosphate groups within the recognition sequence plays a limited role in enzyme inhibition. The presence of multiple dNMPS residues at regions of the DNA removed from the EcoRV recognition site may decrease the amount of enzyme available for catalysis by nonspecific binding to EcoRV.

Base Sequence↗
PubMed1981

Synthesis of nucleoside 3'-(S-alkyl phosphorothioates) and their use as substrates for nucleases.

The synthesis of cytidine, uridine, guanosine, and adenosine 3'-(S-methyl phosphorothioates) by treatment of the 2',5'-di-O-(4-methoxytetrahydropyran-4-yl)ribonucleosides with 2-(methylthio 4H-1,3,2-benzodioxaphosphorin 2-oxide is described. These nucleotide analogues are stable compounds both in the solid state and the neutral aqueous solution. All four of these compounds are degraded by RNase T2 to the parent nucleotides and methanethiol. In addition, cytidine and uridine 3'-(S-methyl phosphorothioates) are substrates for bovine pancreatic ribonuclease and guanosine 3'-(S-methyl phosphorothioate) is a substrate for RNase T1 and RNase U1. When used in conjunction with a chromophore-producing reagent, nucleoside 3'-(S-methyl phosphorothioates) provide a means for direct kinetic measurement of ribonuclease activity over a wide pH range (pH 2-9). The reactivities of these substrates with ribonucleases are compared to the reactivities of other synthetic substrates as well as a number of natural substrates. The utility of ribonucleoside 3'-(S-methyl phosphorothioates) as substrates for the assay of ribonucleases is discussed.

Animals↗
PubMed1977

Incorporation of phosphorothioate groups into fd and phi X174 DNA.

We have synthesized fd and phi X174DNA in the presence of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP alpha S) and the corresponding phosphorothioate derivatives of dCTP and dTTP using ether-permeabilized E. coli cells or crude cell extracts of E. coli DNA polymerase I. Reaction rates of enzymes involved in the formation or breakdown of DNA are decreased in the presence of phosphorothioates. The amount of label incorporated with [35S]dATP alpha S suggests that the dAMP has been completely substituted by 2'-deoxyadenosine 5'-0-phosphorothioate (dAMPS). The substituted DNAs have the same sedimentation coefficients, similar buoyant density, infectivity, and thermal stability as the unsubstituted DNAs. The procedure therefore allows specific modification at the 5' position of dA, dC, or dT in the DNA. In view of the recent demonstration of specific binding of Pt2+ complexes to the phosphorothioate analogue of poly[r(A-U)] (Strothkamp, K.G., and Lippard, S.J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 2536), the synthesis of phosphorothioate containing DNA may be of use for DNA sequencing by electron microscopy.

Adenosine Triphosphate↗