Physicochemical properties of phosphorothioate oligodeoxynucleotides.

We have recently shown that phosphorothioate (PS) oligodeoxynucleotide (ODN) analogs, unlike their normal congeners, exhibit significant anti-HIV activity (Matsukura et al., (1987) Proc. Natl. Acad. Sci. USA 84, 7706-7710). We now report the syntheses, melting temperatures (Tm), and nuclease susceptibilities of a series of phosphorothioate ODN analogs. These include all-PS duplexes, duplexes with one normal chain and the other chain either all-PS, or end-capped with several PS groups at both 3' and 5' ends. The DNase susceptibilities of the S-ODNs are much less than the normal phosphodiesters, but by contrast duplexes of poly-rA with S-dT40 are much more susceptible to RNase H digestion. The Tm's for AT base pairs of S-ODNs are significantly depressed relative to normals, while GC base pairs show much less Tm depression. The Tm's of S-dT oligomers with poly-rA are reduced relative to the duplexes with normal dA oligomers. These results have significance for the biological properties of these analogs as anti-message inhibitors of gene expression, and provide a rational basis for the S-dC/G sequences as potential effective anti-AIDS agents.     

Synthesis and Properties of Novel 5′-Linked Oligos

Abstract

We have synthesized normal and phosphorothioate oligos with 5′-linked groups, using either a phosphoramidite with the linked group attached or a mercaptopropanol linker. These linked oligos have been studied for cellular uptake as fluorescent labels, and for inhibition of gene expression in a cell free expression system, and in several other biological systems.     

Anti-sense phosphorothioate oligonucleotides have both specific and non-specific effects on cells containing human papillomavirus type 16.

A range of specific nuclease resistant phosphorothioate oligodeoxynucleotides (S-oligos) complementary to mRNA of human papillomavirus type 16 (HPV16), were tested for their ability to inhibit cell proliferation and to alter the level of HPV-specific mRNA and proteins in CaSki cells, a human cervical carcinoma cell line containing HPV16 DNA. Only certain of the S-oligos to the viral upstream regulatory region (URR) and the early viral open reading frames (ORF), E6 and E7, were found to display any activity on the cells. These S-oligos were found to exhibit potent anti-proliferative activity at concentrations between 0.25 microM and 20 microM, inhibiting the uptake of [3H]-thymidine into CaSki cells by up to 90% at higher concentrations. The rate of synthesis of E6 and E7 proteins and the steady state level of the E7 protein however remained largely unchanged. E7 protein exhibited a greater decrease in phosphorylation in the presence of only one of the antisense oligos. Other S-oligos including a random sequence, unmodified sequences or O-methylphosphonate modified oligos, had no specific effect on the cells. The results imply that the anti-sense S-oligonucleotides had both specific anti-HPV16 and other non-specific effects on cell proliferation and synthesis of virally encoded proteins.     

Modified oligonucleotides in rabbit reticulocytes: uptake, stability and antisense properties.

We have investigated the behaviour of antisense oligonucleotides in rabbit reticulocytes. Both backbone-modified oligomers (methyl-phosphonate and phosphorothioate analogues), anomers of nucleotide units (alpha) and oligonucleotides linked to various ligands (intercalator, polylysine, dodecanol) were tested with respect to cellular uptake and inhibition of protein synthesis. Oligonucleotides added at an external concentration of 10 microM slowly entered the cell up to a concentration of a few hundred nanomolars. A large fraction of phosphorothioate analogues was found to be associated with the membrane. alpha-, methylphosphonate and phosphorothioate analogues remained intact during the incubation with reticulocytes although the latter were partly dephosphorylated. Antisense oligonucleotides were targeted against three different sites of the rabbit beta-globin mRNA: the 5' end of the message, the initiator AUG or the coding sequence. No specific effect on beta-globin synthesis was observed with any of the investigated compounds.     

Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV)

Nuclease-resistant phosphorothioate analogues of oligodeoxynucleotides (oligos) were synthesized by sulfurization of either internucleoside phosphite linkages, in a repetitive manner during chain extension, or internucleoside hydrogen phosphonate linkages, in a single step following chain assembly. These analogues were tested as antiviral agents against human immunodeficiency virus (HIV). In a cytopathic effect inhibition assay using HIV-uninfected susceptible T cells (tetanus toxoid-specific normal T cells) co-cultured with irradiated chronically HIV-infected cells, phosphorothioate oligomers inhibited the cytopathic effect and replication of several isolates of HIV-1 and HIV-2. Thus phosphorothioate analogues of oligos could inhibit cell-to-cell transmission of the virus as well as the infection by cell-free virus particles and also could inhibit a variety of isolates of human retroviruses.     

Stability of antisense DNA oligodeoxynucleotide analogs in cellular extracts and sera

Antisense DNA oligodeoxynucleotides can selectively inhibit the expression of individual (undesirable) genes and thus, have potential in the treatment of cancer and viral diseases. A prerequisite to their use as therapeutic agents is information on the stability of oligodeoxynucleotides, and their structurally modified analogs, in the biological milieu. To this end, degradation of 5' end and internally [32P] labelled unmodified DNA oligodeoxynucleotide (D-oligo) and analogs containing phosphorothioate (S-oligo), methylphosphonate (MP-oligo), and novel alternating methylphosphonate and phosphodiester (Alt-MP-oligo) internucleoside linkages was studied in Hela cell nuclear extract, S100 cytoplasmic extract, normal human serum and calf serum at 37 degrees C. Both 5' end and internally labelled D-oligos showed complete degradation within 30 min incubation in human serum at 37 degrees C. In any given medium, the D-oligo was the least stable oligodeoxynucleotide to nuclease degradation whereas the Alt-MP, MP and S-oligos were generally of comparable stability and all relatively more stable than D-oligo. Interestingly, MP and Alt-MP-oligos also exhibited greater resistance to phosphatases in cellular extracts compared to D and S-oligos. Under the conditions of the experiments, increasing degradation for any given oligonucleotide was observed in the order: S100 cytoplasmic extract less than nuclear extract less than normal human serum less than calf serum. In a study involving alpha-MEM cell culture medium containing 10% heat inactivated fetal calf serum (heated to 56 degrees C for 1 hour), the D-oligo was found to be rapidly degraded (degradation evident within 10 mins) whereas degradation products for the S-oligo were observed within 1 hour. In contrast, the Alt-MP oligo remained stable throughout the 3 hour experiment. These results indicated that in cell culture medium containing heat inactivated serum Alt-MP oligo was more stable than D- and S-oligos.     

Downregulation of the p75 neurotrophin receptor in tissue culture and in vivo, using beta-cyclodextrin-adamantane-oligonucleotide conjugates

Formation of complexes with beta-cyclodextrin derivatives via adamantyl groups was found to enhance the uptake and antisense efficacy of phosphorothioate oligos targeted to the p75 neurotrophin receptor in neuronally differentiated PC12 cells. After a 2-week course of systemic administration to mice (by intraperitoneal injection), there was evidence of a pronounced uptake of these oligos by the dorsal root ganglia (DRG), as well as by liver and kidney. There was no uptake by the brain. Consistent with uptake of antisense oligos by the DRG, systemic administration resulted in marked and consistent downregulation of p75 in DRG neurons. These results indicate that cyclodextrin-adamantane-oligo conjugates have great potential as agents to downregulate target genes in neurons, particularly in vivo in the peripheral nervous system.     

Antisense inhibitory effect: a comparison between 3'-partial and full phosphorothioate antisense oligonucleotides.

Phosphorothioate (PS) antisense oligonucleotides are currently used to inhibit many cell functions both in vivo and in vitro. However, these modified oligos provide reasonable sequence specificity only within a narrow concentration range. To overcome such a limitation we synthesized antisense oligomers, partially phosphorothioated, targeted against the human N-myc mRNA. We utilized such modified oligomers in a human neuroblastoma cell line where the N-myc gene expression was very high, and compared them to full phosphorothioate oligonucleotides. Both full PS and partial PS antisense oligos produced a maximum reduction in target mRNA after 6 h of treatment. They were able to maintain a good level of inhibition for 20 h only at high concentration. While partial PS oligos produced a dose dependent and sequence specific inhibition of N-myc mRNA, full PS molecules suffer from some disadvantages at the highest concentration used. Our results showed that partial PS molecules were capable of reducing gene expression showing a greater sequence specificity over a far broader concentration range. For this reason we conclude that partial PS antisense oligos, with respect to full PS antisense oligos, might be particularly useful for studying gene function.     

Enhancing the stability of oligonucleotide duplexes. The effect of adding 1-(2'-deoxy-beta-d-ribofuranosyl)-5-nitroindole

We studied the properties of DNA duplexes containing 5-nitroindole (N) in one of the chains. We synthesized 8-membered oligos with N at the 5' or at the 3' end: 5'-d(NXGACCGTC)-3' or 5'-d(GACCGTCXN)-3', where X is one of the four natural bases, making all four kinds of oligos with and without N. We also prepared 11-membered oligos complementary to the above octanucleotides: 5'-d(TGACGGTCYZT)-3' and 5'-d(TZYGACGGTCT)-3', where Y and Z are A, G, C, or T. The stability of duplexes obtained with these oligos was assessed by melting, and the thermodynamic parameters delta H, delta S, and Tm were calculated. Comparison of the melting curves for modified and nonmodified duplexes demonstrated that the presence of N at the 5' end of one chain raises the Tm by 6.6 degrees C on average; if N is at the 3' end of the same chain, the Tm increases by about 3 degrees C.     

Patterns of intracellular compartmentalization, trafficking and acidification of 5'-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells.

We have examined the intracellular compartmentalization and trafficking of fluorescein labeled (F) phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos) in HL60 cells. A series of F-oligos (PO and PS) were incubated for 6 hrs. with HL60 cells and the mean intracellular fluorescence determined by flow cytometry. The F signal was normalized by the addition of the ionophore monensin. An increase in signal intensity following addition of monensin indicated that the oligo was resident in an acidic intracellular environment. F-PS, but not F-PO oligos were found to reside in an acidic environment. An exception was a PO homopolymer of 15 cytidine bases (FOdC15) which was acidified. Using two different methods, the average resident intracellular pH of F-PS oligos and F-OdC15 was shown to be approximately 1 pH unit lower than that of F-PO oligos. Acidification of F-PS oligos could be blocked by the antibiotic bafilomycin, indicating that acidification was occurring in endosomes or vacuoles. F-PO and F-PS oligos were effluxed from HL60 cells from two intracellular compartments. However, approximately 60% of internalized F-PO oligo resided in a 'shallow' compartment that was turned over rapidly (t1/2 = 5-10 min.) whereas only 20% of F-PS oligo resided in this compartment. Conversely, approximately 80% of the internalized F-PS oligo but only 40% of F-PO oligo resided in a 'deep' compartment that turned over with t1/2 = 2-5 hrs. This report is the first quantitative demonstration that PO and PS oligos, and PO oligos of different sequences are trafficked differently by HL60 cells.     

Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression--a review

Synthetic oligodeoxyribonucleotides (oligos) are now widely used as artificial regulators for gene expression both in cell-free media and in cultured cells. We describe the biological consequence of the various chemical modifications that have been introduced into the molecules to improve their resistance against nuclease attack, their affinity for the target mRNA and their uptake by cells. We also describe the rising generation of antimessenger oligos. Covalently linked to reactive groups these molecules direct irreversible modifications of the complementary nucleic acids. We anticipate that these oligos will be targeted to double-stranded nucleic acids to interfere with gene expression at the DNA level.     

Pharmacokinetics,Tissue Distribution,and Safety of P-Ethoxy Oligonucleotides Incorporated in Liposomes

Abstract

P-ethoxy oligonucleotides (oligos) are lipophilic analogs of phospho-diesters. We have used liposomes to increase the intracellular uptake of P-ethoxy oligos, and demonstrated that liposomal P-ethoxy antisense oligos specific for Bcr-Abl, Grb2, Crkl or Bcl-2 mRNA could selectively inhibit the production of the corresponding proteins, thereby inducing growth inhibition in leukemia and lymphoma cell lines. In support of studying the effectiveness of liposomal P-ethoxy antisense oligos in animal models, we had conducted a series of studies to evaluate the pharmacokinetics, tissue distribution and safety of intravenous injection of liposomal P-ethoxy oligos in normal mice. The pharmacokinetics and tissue distribution of liposomal P-ethoxy oligos are very similar to those of other liposomal compounds. The plasma clearance rate of liposomal P-ethoxy oligos was biphasic; the t_1/2 α and t_1/2 β were approximately 6.7 min and 7 h, respectively. The highest concentrations of liposomal P-ethoxy oligos were found in spleen and liver, with a t_1/2 of approximately 48 h. When up to 180 mg of P-ethoxy oligos per kg of mice's body weight were used, the administration of liposomal P-ethoxy oligos had no adverse effects on renal and hepatic functions, or on the hematological parameters studied. No major organ pathologic changes were observed. Our studies suggested that, at the doses studied, liposomal P-ethoxy oligos could be safely used in animal studies. Since liposomal P-ethoxy oligos were found to accumulate mainly in spleen and liver, which are the major organs of leukemic and lymphoma disease manifestation, we are currently investigating the use of liposomal P-ethoxy antisense oligos in experimental leukemia and lymphoma animal models.     

Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity.

We have studied factors which may effect the intracellular availability of oligonucleotides to achieve antisense activity. 15-20 mer unmodified, phosphorothioate modified and liposomally encapsulated oligodeoxynucleotides have been tested in leukemia MOLT-3 cells. Phosphorothioate analogs penetrated and accumulated intact in cells in contrast to unmodified oligomers, which showed a high instability in cell culture medium. A slow decrease of intracellular concentration of undegraded phosphorothioate oligodeoxynucleotides was observed after cell treatment and could be predominantly explained by a significant efflux transport. Using laser-assisted confocal microscopy we have observed that fluorescein 5-end-labeled phosphorothioate derivatives predominantly distributed in intracytoplasmic endocytic vesicles following cell treatment. The end-capped version of phosphorothioate oligodeoxynucleotides exhibited greater cellular uptake than fully modified analogues while exhibiting similar biological stability. Liposome encapsulation made possible oligomer protection in serum-containing medium and substantially improved cellular accumulation. Furthermore, the efflux rate of oligomer initially introduced within liposomes is 2-fold lower than that observed in cells which have been incubated with free oligonucleotides. Liposomal preparations of oligodeoxynucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. Furthermore, intracellular distribution studies demonstrate that intracellular transport of unmodified oligomers is effectively achieved using the liposomal carrier.     

Diacridines: bifunctional intercalators. I. Chemistry, physical chemistry and growth inhibitory properties.

The synthesis, as well as the rationale for synthesis of diacridines, double intercalators, as potential inhibitors of nucleic acid synthesis is presented. The syntheses of (9-acridyl)-putrescine and -spermine, and bis(-9-acridyl)-putrescine, -spermidine, -spermine diamines and of bis(6-chloro-2-methoxy-9-acridyl)-putrescine and -spermine diamines, all substituted on the terminal NH2 groups are described. In addition, the homologous series of diacridines connected by the amino groups of the diamines NH2(CH2)nNH2 (where n = 2,3,4,6,8,10,12,14,16,18) to the C-9 of the diacridines has been synthesized. The chemical properties of these compounds as well as their molecular relationship to DNA are presented. The effect of the double intercalators on the Tm of DNA and of (A)n - (U)n, (dA)n - (dT)n, (G)n - (C)n and on (dG)n - (dC)n have been determined. The double acridine intercalators produce a much greater increase of the Tm of these nucleic acids than do the single acridine intercalators. They also profoundly affect the Tm of DNA in physiological salt concentrations; under these latter conditions the single intercalators have no effect. The relationship between the length of the chain connecting the two acridine rings and the inhibition of the growth of P-388 cells in vitro and vivo is presented. Their growth inhibitory properties appear, in general, to parallel their intercalative abilities.     

Phosphorothioate analogues of oligodeoxyribonucleotide: synthesis and activity as inhibitors of replication of human immunodeficiency virus

The modifications of oligodeoxyribonucleotides include replacement of the other chain either all-PS (S-ODNs), or end-capped with several PS (SO-ODNs) groups at both 3'- and 5'-ends. A general synthesis of phosphorothioate analogues of oligodeoxyribonucleotides is described using the new phosphite. In assays of HIV, oligomers (S-ODNs) with complete replacement of phosphodiesters with phosphorothioate groups were found to be very active. Finally of particular interest is S-ODNs-rev or tat (20mers) which possessed slightly higher anti-HIV activity than S-dC28 itself. By contrast, the unmodified oligomers (ODNs) as well as SO-ODNs did not have any inhibitory effect.     

Rapid nuclear accumulation of injected oligodeoxyribonucleotides.

The intracellular transport and fate of nucleic acids is poorly understood. To study this process, we injected fluorescent oligodeoxyribonucleotides (oligos) into the cytoplasm of CV-1 epithelial cells and primary human fibroblasts. Rapid nuclear accumulation was found with the phosphodiester (PD), phosphorothioate (PT), and methylphosphonate (MP) forms of a 28-mer oligo complimentary to the rev mRNA of the human immunodeficiency virus type 1. Migration of the oligos in the cytoplasm was slower than diffusion of a coinjected dextran, but the oligos freely diffused into the nucleus. Nuclear incorporation was temperature but not energy dependent. The intranuclear distribution of the oligos was influenced by the chemistry of internucleoside linkages. The PD oligos and, to a lesser extent, the PT oligos colocalized with small nuclear ribonucleoproteins (snRNPs), whereas the MP oligos colocalized with concentrated regions of genomic DNA. These data have important implications for our understanding of the transport and accumulation of exogenous nucleic acids in mammalian nuclei, and the assay described could potentially be used for testing the efficacy of oligos designed as therapeutic agents.     

Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA

The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.     

Kinetics and thermodynamics of i-DNA formation: phosphodiester versus modified oligodeoxynucleotides.

At slightly acidic or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines have been shown to form a tetrameric structure in which two parallel-stranded duplexes have their hemiprotonated C.C+base pairs face to face and fully intercalated, in a so-called i-motif. Cytosine-rich pyrimidine oligodeoxynucleotides can form an intramolecular i-motif. We have studied the ability of several DNA analogs to fold into this structure. Evidence for folding was provided by thermal denaturation. We have shown that phosphorothioate and phosphodiester oligodeoxynucleotides, but not methylphosphonate or PNA oligomers, may form the i-motif. Four different PS oligodeoxynucleotides were compared with their PO counterparts. In all cases, the melting temperature (Tm) of the phosphorothioate oligomer was equal or slightly inferior (by 2-3 degreesC) to the Tmof the natural oligodeoxynucleotide. For long oligodeoxynucleotides, a small change of pH leads to a completely different melting profile: the curves are reversible at pH 6.4 or lower, and a hysteresis is obtained at pH 6.8 or higher; cooling and heating curves were not superimposed, allowing us to determine the rate constants of association (kon) and dissociation (koff) as a function of the temperature: these rate constants give linear Arrhenius plots, in agreement with the prediction of the two-state model of association-dissociation. The activation energy Eonis strongly negative and, at neutral pH, the phosphorothioate associates and dissociates nine times faster than the phosphodiester oligodeoxynucleotide of identical sequence.     

Conjugation reactions involving maleimides and phosphorothioate oligonucleotides

Phosphorothioate diester oligonucleotides proved to be fully compatible with maleimides in the context of two different conjugation reactions: (a) reaction of (5')diene-[phosphorothioate oligonucleotides] with maleimido-containing compounds to afford the Diels-Alder cycloadduct; (b) conjugation of (5')maleimido-[phosphorothioate oligonucleotides] with thiol-containing compounds. No evidence of reaction between phosphorothioate diesters and maleimides was found in any of these processes. Importantly, in the preparation of (5')maleimido-[phosphorothioate oligonucleotides] from [protected maleimido]-[phosphorothioate oligonucleotides], which requires the maleimide to be deprotected by retro-Diels-Alder reaction (heating for 3-4 h in toluene at 90 °C), no addition of phosphorothioate diester to the maleimide was found either. Finally, maleimide-[phosphorothioate monoester] conjugation was also explored for comparison purposes.