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PubMed1987

DNA and RNA sequencing utilizing phosphorothioate chemistry.

A method for nucleic acid sequencing has been developed based on the observation that phosphorothioate diesters are hydrolysed by treatment with 2-iodoethanol in a solution of aqueous ethanol. For DNA sequencing, primed single-stranded M13 DNA is polymerised with the Klenow fragment of DNA polymerase I in the presence of the three normal deoxyribonucleotide triphosphates and one alpha-phosphorothioate derivative. This is followed by treatment with 2-iodoethanol, precipitation of the DNA fragments and analysis by polyacrylamide electrophoresis. RNA transcribed from plasmids containing the SP6 RNA polymerase promoter is sequenced by including the alpha-phosphorothioate derivative of the ribonucleotide triphosphates in the polymerisation and treating the product with iodoethane. The cleavage reaction involves alkylation of the sulfur atom to form the phosphorothioate triester and hydrolysis catalysed by an adjacent hydroxyl group.

Base Sequence↗

PubMed1987

Self-splicing of Tetrahymena rRNA can proceed with phosphorothioate substitution at the splice sites.

The self-excision of a 413-base intervening sequence of the 26S rRNA of Tetrahymena thermophila has been investigated using phosphorothioate-substituted RNA. Transcripts containing this intron were prepared by T7 RNA polymerase-catalyzed polymerisation using a M13 mICE10 vector in the presence of various nucleoside alpha-thiotriphosphate analogues. Wild-type transcripts incorporating phosphorothioates 5' to adenosine or uridine were inactive, whereas incorporation 5' to cytidine or guanosine allowed splicing. The first two substitutions place phosphorothioates inter alia at the 5' and 3' splice sites respectively. Mutagenesis at either site allowed phosphorothioate substitution 5' to guanosine at each splice site. This did not block splicing, suggesting that substitution at internal sites within the intron has more effect.

Animals↗

PubMed1994

Pharmacokinetics, tissue distribution, and stability of antisense oligodeoxynucleotide phosphorothioate ISIS 3466 in mice.

Phosphorothioate oligonucleotides have a potential as therapeutic agents. The pharmacokinetics, tissue distribution, stability, and cellular uptake by LOX ascites tumor of p120 antisense phosphorothioate oligonucleotide, ISIS 3466, were studied in vivo. The oligonucleotide, which was quickly cleared from the circulation in the normal mice after IV injection, was readily absorbed into the systemic circulation from the peritoneum. The oligonucleotide was found in most tissues 48 h after IP administration. The highest concentrations were in kidney and liver, but the brain had a very low concentration. The phosphorothioate oligonucleotide was intact even after 48 h. When the oligonucleotide was complexed with cationic lipid DOTMA, the DOTMA did not affect the oligonucleotide uptake or tissue distribution in normal mice. However, DOTMA significantly increased the oligonucleotide cellular uptake (4-10 times) in LOX ascites tumors in an IP/IP model. These results indicate that the phosphorothioate oligonucleotide is stable, has favourable kinetics for use as an therapeutic agent, and that DOTMA could be useful in local delivery of the oligonucleotide in vivo.

Animals↗

PubMed1994

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate.

Biodistribution and metabolism of oligonucleotides were determined using a 3H-labeled 20-nucleotide phosphodiester and its phosphorothioate analog. The oligonucleotides were radiolabeled by 3H-methylation of an internal deoxyctidine with HhaI methylase and S- [3H]adenosylmethionine. Biodistribution studies were conducted after intravenous injection of 6 mg/kg (5 muCi) oligonucleotide. Metabolism of the oligonucleotides was determined by paired-ion high performance liquid chromatography. After phosphodiester injections, radiolabel rapidly cleared the blood. Relative initial concentrations were as follows: kidney > blood > heart > liver > lung > spleen. Radiolabel in spleen peaked at 1 hr and remained elevated for 24 hr. At 2 hr the concentration in all organs, except spleen, was equal to that in blood. High performance liquid chromatographic analysis of the kidney, liver, and spleen extracts and urine indicated extremely rapid metabolism to monomer. Results of studies after the injection of phosphorothioate oligonucleotide differed from those using the phosphodiester. Despite its rapid clearance from blood, phosphorothioate accumulated rapidly in all tissues, especially the kidney. Kidney uptake increased over time, remaining very high for 24 hr. Ratios of organ to blood concentrations at 2 hr for all organs were 5:1 or greater. Kidney and liver ratios were 84:1 and 20:1, respectively. Analysis of the kidney and liver extracts and urine indicated that slow metabolism occurred. These data suggest that phosphodiester oligonucleotides would have limited therapeutic utility. The stability and organ distribution of the phosphorothioate oligonucleotide imply that such oligonucleotides may have therapeutic potential.

Animals↗

PubMed1996

Comparative properties of a technetium-99m-labeled single-stranded natural DNA and a phosphorothioate derivative in vitro and in mice.

Oligonucleotides, particularly single stranded, may ultimately be of considerable use as radiopharmaceuticals. We have compared a synthetic 22-base single-stranded phosphodiester DNA with its phosphorothioate analog after both were radiolabeled with 99mTc via the hydrazino nicotinamide chelator. Whole body clearance of the label in mice was much slower when introduced on the phosphorothioate (30% vs. 75% clearance at 6 hr) because of immediate and persistent accumulation in liver (47% vs. 2% injected dose/g at 4 hr). The label in both cases was present in urine primarily on low molecular weight catabolites. High-performance liquid chromatography analysis of 37 degrees C serum incubates showed serum protein binding of 99mTc in both cases (about 100% bound at 24 hr) but to different proteins. Different behavior with respect to protein binding was also observed in the analysis of liver and kidney homogenates: the phosphodiester label was almost quantitatively converted to lower molecular weight catabolites after only 15 min, whereas the phosphorothioate label was primarily on proteins. The rapid digestion of the phosphodiester by nucleases was not observed, probably because protein binding of the labeled oligonucleotides stabilized against degradation. Thus the phosphodiester DNA may be the preferred 99mTc-labeled oligonucleotide in certain circumstances to avoid the high and persistent liver uptake observed with the phosphorothioate DNA.

Animals↗

PubMedDate not supplied

Phosphorothioate substrates for the SfiI restriction endonuclease.

Oligodeoxynucleotides carrying the recognition sequence for the SfiI endonuclease were synthesised with phosphorothioates at the cleavage site. The Rp and Sp diastereoisomers of the oligonucleotides were separated by HPLC using a mobile phase containing L-cysteine. The duplex with Rp phosphorothioates was cleaved very slowly in the presence of Mg2+, though virtually complete cleavage was obtained with Mn2+. No significant cleavage of the duplex with Sp phosphorothioates occurred with either Mg2+ or Mn2+. When added to a plasmid with one SfiI site, the duplexes with either Rp or Sp phosphorothioates inhibited the rate at which SfiI cleaved the plasmid: a control duplex with oxyester linkages enhanced the rate of plasmid cleavage. In contrast to type IIe nucleases such as EcoRII and NaeI, which can be activated by non-hydrolysable analogues of their substrates, SfiI reactions require four susceptible phosphodiester bonds.

Deoxyribonucleases, Type II Site-Specific↗

PubMed2002

Complement activation is responsible for acute toxicities in rhesus monkeys treated with a phosphorothioate oligodeoxynucleotide.

The objective of this study was to define the role of complement activation in the acute and transient toxicities associated with administration of phosphorothioate oligonucleotides in monkeys. In the absence of complement inhibitor, complement activation blocker-2 (CAB-2), i.v. infusion of 20 mg/kg ISIS 2302 produced increases in the concentrations of the complement split products Bb and C5a (100- and 7-fold, respectively). Monkeys also experienced marked changes in bloodpressure (hypertension and hypotension), clinical signs of toxicity (lethargy and periorbital edema), fluctuations in circulating neutrophil counts, and elevations in serum cytokine levels (45-, 12-, and 4-fold increases in IL-6, MCP-1, and IL-12, respectively). Changes occurred at or near the end of infusion and returned to normal over time. One of the three animals died approximately 4 h following infusion of 20 mg/kg ISIS 2302 alone. In contrast, prior treatment with CAB-2 effectively blocked complement activation, as well as the ISIS 2302-induced hemodynamic and clinical responses. Importantly, plasma concentration of ISIS 2302 were unaffected by CAB-2 pretreatment. Thus, the protection afforded by CAB-2 was due to its inhibition of complement activation rather than to any impact on the disposition of ISIS 2302. These results clearly demonstrate the causal relationship between activation of the alternative complement pathway and the hemodynamic and clinical responses associated with rapid infusion of phosphorothioate oligonucleotides. Demonstration of this relationship underscores the importance of avoiding complement activation in patients to ensure the continued safe use of phosphorothioate oligodeoxynucleotides.

Animals↗

PubMed2003

[Effect of 3' exonuclease activity of polymerase on extension of phosphorothioate-modified primers].

OBJECTIVE: To determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase. METHODS: Two-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated. RESULTS: Exo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase. CONCLUSION: These data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

DNA Primers↗

PubMed1998

Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex.

Systemic administration of ISIS 2302, a 20-mer antisense phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both monkey and human studies. The anticoagulant effects of ISIS 2302 were investigated with both in vitro coagulation assays in human plasma and purified enzyme systems. At high oligonucleotide plasma concentrations (>100 microgram/mL), prolongation of the prothrombin and thrombin times was observed. In a thrombin time assay using purified components, high concentrations of ISIS 2302 inhibited thrombin clotting activity both by stimulating inhibition by heparin cofactor II and directly competing with fibrinogen for binding to anion binding exosite I. In contrast, low concentrations of ISIS 2302 (<100 microgram/mL) showed a selective, linear prolongation of the activated partial thromboplastin time (PTT). The rate limiting effect of 50 microgram/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was identified by sequential modification of the clotting assay. Delaying addition of oligonucleotide until after contact activation failed to correct prolongation of the PTT. The calcium-dependent steps of the intrinsic pathway were individually assessed by adding sufficient activated coagulation factor to correct the PTT in plasma deficient in that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor X-deficient plasma with or without oligonucleotide present. ISIS 2302 (50 microgram/mL) did not prolong a modified Russel viper venom time, suggesting no significant inhibition of prothrombinase. Thus, 50 microgram/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic tenase activity. ISIS 2302 showed partial inhibition of intrinsic tenase activity (to approximately 35% of control) at clinically relevant oligonucleotide concentrations in a chromogenic assay. This activity was oligonucleotide sequence-independent but required the phosphorothioate backbone, suggesting that inhibition of intrinsic tenase is a general property of this class of oligonucleotides. These results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.

Animals↗

PubMed1995

Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro.

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.

Animals↗

PubMed1993

Method for sequencing synthetic oligodeoxynucleotide phosphorothioates.

Sequencing of oligodeoxynucleotide phosphorothioate by a modified Sanger method of sequencing is described. The procedure involves ligation of synthetic oligodeoxynucleotide phosphorothioate to an oligodeoxynucleotide, referred to here as "helper oligonucleotide." The helper oligonucleotide has a region which is complementary to T7 primer. By using DNA polymerase and nucleoside triphosphate mixture, 5'-labeled T7 primer is extended onto ligated oligodeoxynucleotide phosphorothioate, which is then analyzed on gel electrophoresis.

Base Sequence↗

PubMed1996

Quantitation of phosphorothioate oligonucleotides in human plasma.

Methods are presented for the extraction of phosphorothioate oligonucleotides from human plasma to permit quantitation by capillary gel electrophoresis. Extraction of the phosphorothioate oligonucleotides from plasma was accomplished using two solid-phase extraction columns, a strong anion-exchange column to remove plasma proteins and lipids, followed by a reverse-phase column to remove salts. A second desalting step, achieved by dialysis utilizing a membrane with a molecular weight cutoff of 2500 Da floating on distilled water, was required to remove residual ionic material from the extracted sample. This method should be generally applicable to the analysis and quantitation of phosphorothioate oligonucleotides.

Electrophoresis, Capillary↗

PubMed1995

Phosphorothioate oligonucleotides block reverse transcription by the Rnase H activity associated with the HIV-1 polymerase.

We demonstrate the degradation of RNA bound to an antisense oligonucleotide by a reverse transcriptase enzyme-associated RNase H activity. We found that phosphorothioate oligonucleotides inhibit the RNase H activity by binding to AMV RT, rather than to the template RNA, whereas the RNase H activity of HIV-1 RT is not affected by the antisense phosphorothioate oligonucleotide. Selective inhibition of HIV-1 gene expression involves the degradation of the template RNA bound to the antisense phosphorothioate oligonucleotide by the RNase H activity associated with the HIV-1 polymerase.

Avian Myeloblastosis Virus↗

PubMed1996

Anti-sense oligodeoxynucleoside phosphorothioates nonspecifically inhibit invasion of red blood cells by malaria parasites.

Anti-sense and sense oligodeoxynucleoside phosphorothioates, based on analysis of the secondary structure of Plasmodium falciparumdihydrofolate reductase-thymidylate synthase mRNA, were synthesized. Their effects on P. falciparum growth in vitro were examined by microscopy and [3H] hypoxanthine incorporation. Both anti-sense and sense oligodeoxynucleoside phosphorothioates inhibit invasion of red blood cells by merozoites and this is interpreted as being caused by their polyanionic nature. Specific anti-sense effects of the oligonucleoside phosphorothioates could not however be demonstrated.

Animals↗

PubMed1996

Inhibition of cytochrome P450 1A1 by antisense phosphorothioate oligonucleotide in Hepa lclc7 cells.

The influence of an antisense phosphorothioate oligonucleotide has been investigated on 7-ethoxyresorufin O-deethylase (EROD) activity and CYP1A1 protein in wild type mouse hepatoma Hepa lclc7 (Hepa-1) cells. The results show that administration of a 15-mer antisense phosphorothioate oligonucleotide in ribonucleoside-free minimum essential medium effectively inhibited UV-oxidized tryptophan-inducible EROD activity and CYP1A1 protein. The inhibition of EROD activity was dose- and time-dependent. The inhibition of oxidized tryptophan-inducible EROD activity after administration of 5 microM antisense oligonucleotide for 18 hours was 74% over the control oligonucleotide-administered cells. There was no effect of the control or antisense oligonucleotide on the cell growth. This is the first demonstration that inducible CYP1A1 can be effectively inhibited by antisense phosphorothioate oligonucleotide in Hepa-1 cells. Utility of this approach should be useful in elucidating the role(s) of CYP1A1 in chemical carcinogenesis.

Animals↗

PubMed1997

Inhibition of HIV-1 replication by triple-helix-forming phosphorothioate oligonucleotides targeted to the polypurine tract.

We show the effects of triple-helix formation by assays of primer extension inhibition in vitro using two systems (two-strand-system (FTFOs) or three-strand-system (TFOs) targeted to the polypurine tract (PPT) of HIV-1. The FTFOs were more effective than the TFOs. We found that the FTFOs containing phosphorothioate groups at the 3'- and 5'-ends, or inside the hairpin loop, exhibited higher inhibitory effects on cDNA synthesis and greater exonuclease resistance than the unmodified FTFOs and TFOs. The abilities of the FTFOs containing phosphorothioate groups at the antisense sequence sites to inhibit HIV-1 replications were examined. The FTFOs containing phosphorothioate groups at the antisense sequence sites inhibit the replication of HIV-1 more efficiently than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.

Anti-HIV Agents↗

PubMed2000

Stimulation of thymocyte proliferation by phosphorothioate DNA oligonucleotides.

DNA is a complex macromolecule the immunological properties of which depend on short sequence motifs called CpG motifs or immunostimulatory sequences (ISS). These sequences are mitogenic for B cells and can stimulate macrophage cytokine production. While these sequences do not directly activate T cells, they can augment effects of stimulation via the TCR. Furthermore, ISS can affect T cells because of macrophage production of IL-12 and IFN-alpha/beta. In these studies, we further evaluated the immune effects of DNA on T cells, testing the possibility that certain T cell populations can respond directly to this stimulus. We therefore tested the in vitro responses of thymocytes to a series of phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) varying in sequence. In in vitro cultures, phosphorothioate ODNs (sODNs) containing CpG motifs induced significant proliferation of murine thymocytes, although phosphodiester compounds lacked activity. The magnitude of stimulation varied with sequences flanking the CpG motifs, as both dA and dT sequences enhanced the stimulatory capacity of the CpG motif. Furthermore, CpG sODNs were strong costimulators of anti-CD3-mediated thymocyte activation, increasing proliferation compared to anti-CD3 in the absence of DNA. This activation was only partially inhibited by cyclosporine A and was not dependent on a calcium influx. Together, these results indicate that phosphorothioate oligonucleotides containing CpG motifs can directly induce thymocyte proliferation as well as augment TCR activation. These observations thus extend the range of actions of CpG DNA and suggest additional mechanisms for its function as an immunomodulatory agent or adjuvant.

Adjuvants, Immunologic↗

PubMed1999

Structural effect of complete [Rp]-phosphorothioate and phosphorodithioate substitutions in the DNA strand of a model antisense inhibitor-target RNA complex.

Chemically modified DNA oligonucleotides have been crucial to the success of antisense therapeutics. Although such modifications are ubiquitous in the clinic, high-resolution structural studies of pharmaceutically relevant derivatives have been limited to only a few molecules. We have completed a high-resolution NMR structural study of three DNA.RNA hybrids with the sequence d(CCTATAATCC). r(GGAUUAUAGG). All hybrids contain an unmodified RNA strand, whereas the DNA strand of each hybrid contains one of three different sugar-phosphate backbone linkages at each nucleotide: (1) phosphate, (2) [Rp]-phosphorothioate, or (3) phosphorodithioate. The UV and NMR melting profiles revealed that the normal hybrid is more stable than the [Rp]-phosphorothioate, which in turn is more stable than the phosphorodithioate. Homonuclear two-dimensional nuclear Overhauser effect spectroscopy and double quantum-filtered correlation spectroscopy afforded nearly complete non-labile proton assignments. The three molecules show nearly equivalent chemical shifts, with the exception of H3' protons, which are shifted downfield in a manner that appears correlated with the degree of sulfur substitution at phosphate. All three hybrids exhibit unusually broad linewidths for deoxyribose protons H2' and H2".Distance restraints were calculated from NOE cross-peak intensities via a complete relaxation matrix approach using the program RANDMARDI. Detailed comparison of interproton distances from each hybrid indicates that the three molecules share a common structure, with neither strand in canonical A or B form. Correlation of R factors, calculated using the program CORMA with DNA H2'-base and H3'-base distances, revealed a relative increase in the population of B-type sugar conformations for deoxyriboses in the A+T-rich center of the hybrid sequence. It is widely known that the activity of enzymes which act upon DNA.RNA hybrid substrates (e.g. ribonuclease H) is impacted when the hybrids contain phosphorothioate or phosphorodithioate substitutions. The structural similarity of the three hybrids examined here suggests that factors other than global structure may mediate the activity of these enzymes.

Base Sequence↗