EP0807171A1 - Synthese d'oligonucleotides de phosphorothioates stereospecifiques - Google Patents

Synthese d'oligonucleotides de phosphorothioates stereospecifiques

Info

Publication number
EP0807171A1
EP0807171A1 EP95943748A EP95943748A EP0807171A1 EP 0807171 A1 EP0807171 A1 EP 0807171A1 EP 95943748 A EP95943748 A EP 95943748A EP 95943748 A EP95943748 A EP 95943748A EP 0807171 A1 EP0807171 A1 EP 0807171A1
Authority
EP
European Patent Office
Prior art keywords
primer
oligonucleotide
rnase
oligonucleotides
template
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP95943748A
Other languages
German (de)
English (en)
Inventor
Jinyan Tang
Allysen M. Roskey
Sudhir Agrawal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aceragen Inc
Original Assignee
Hybridon Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hybridon Inc filed Critical Hybridon Inc
Publication of EP0807171A1 publication Critical patent/EP0807171A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1132—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • This invention relates to antisense oligonucleotides. More specifically, this invention relates to methods of preparing stereospecific phosphorothioate oligonucleotides.
  • PS oligonucleotides Phosphorothioate analogs of oligodeoxynucleotides (PS oligonucleotides) are known to be useful as antisense tools (see, e.g., Agrawal et al. (1989) Proc. Natl. Acad. Sci. (USA)
  • PS oligonucleotides have been chemically synthesized using either the phosphoramidite or the H-phosphonate approach (both reviewed in Methods in Molecular Biology (Agrawal, ed.) Volume 20, Humana Press, (1993) Totowa, NJ) .
  • Such preparative methods introduce a chiral center at each internucleotide linkage that can lead to the formation of 2 n diastereomers per n internucleotide linkages, and generally result in a mixture of about 40% Rp and about 60% Sp diastereoisomers.
  • Rp and Sp diastereosomers may have different biophysical properties, such as different affinities for single-stranded and double-stranded nucleic acids (see, e.g., Cossick et al. (1985) Biochem. 24:3630-3638; Kim et al. (1992) FEBS Lett. 314:29- 32) . Hence, it is desirable under certain circumstances to prepare only one diastereomer.
  • This invention provides, in one aspect, a method of synthesizing the stereospecific Rp diastereomer or isomer of a phosphorothioate oligonucleotide.
  • phosphorothioate oligonucleotide or "PS oligonucleotide” is intended to encompass at least five nucleotides covalently linked 3' to 5' via phosphorothioate internucleotide linkages, and including both Sp and Rp isomers.
  • PS-Rp oligonucleotide refer to only the stereospecific (Rp) isomer of a PS oligonucleotide.
  • a nucleotidic primer is annealed to a complementary oligodeoxynucleotide template having 5' and 3' termini, thereby forming a partially double- stranded/partially single-stranded structure.
  • the primer also has 3' and 5' termini and includes a nucleic acid sequence complementary to a 3' portion of the template.
  • the primer comprises a plurality of deoxyribonucleotides and a ribonucleotide at its 3' terminal or 3' penultimate position.
  • the partially double-stranded/partially single-stranded structure is contacted with a mixture of deoxynucleoside ⁇ -triphosphate Sp isomers and a DNA polymerase for a time necessary and under conditions conducive for the enzymatic synthesis of a PS-Rp oligodeoxynucleotide complementary to a 5' portion of the template, thereby forming a double-stranded product.
  • the deoxynucleoside ⁇ - triphosphate Sp isomers are selected from the group consisting of deoxyguanosine 5'-(c_-thio) triphosphate, deoxyadenosine 5'-( ⁇ .-thio) triphosphate, deoxycytidine 5'-( ⁇ .-thio) triphosphate, and thymidine 5'-( ⁇ -thio) triphosphate.
  • DNA polymerase refers to any enzyme able to covalently link the 3' end of one deoxyribonucleoside to the 5' end of another deoxyribonucleoside, thereby synthesizing DNA. In specific embodiments, synthesis is accomplished with DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, or reverse transcriptase.
  • the double-stranded product is then cleaved after the ribonucleotide at the RNA/DNA junction such that the PS-Rp oligodeoxynucleotide is liberated from its primer and template.
  • cleavage occurs between the primer and the newly synthesized PS-Rp oligonucleotide when the ribonucleotide in the primer is at the 3' terminal position of the primer.
  • cleavage occurs between the 3' penultimate and 3' terminal nucleotide of the primer which has been extended as the PS-Rp oligonucleotide.
  • cleavage is carried out with an endonucleolytic enzyme such as RNase A, RNase Tl, RNase T2, RNase U2, RNase Nl, and RNase N2, while in other embodiments, cleavage is achieved with an alkaline.
  • an endonucleolytic enzyme such as RNase A, RNase Tl, RNase T2, RNase U2, RNase Nl, and RNase N2
  • cleavage is achieved with an alkaline.
  • a strong alkaline or base such as a halide hydroxide selected from the group consisting of sodium hydroxide, ammonium hydroxide, lithium hydroxide, or potassium hydroxide, is used for this purpose.
  • Another aspect of the invention are stereospecific Rp oligonucleotides prepared according to the method of the invention.
  • Yet another aspect of the invention includes an oligonucleotide comprising a plurality of deoxyribonucleotides covalently linked with at least one stereospecific Rp phosphorothioate internucleotide linkage.
  • stereospecific Rp phosphorothioate oligonucleotides are believed to be less mitogenic than Sp phosphorothioates or than a mixture of Rp and Sp phosphorothioates.
  • all of the deoxyribonucleotides are linked with stereospecific Rp phosphorothioate internucleotide linkages.
  • FIG. 1A is a diagrammatic representation of one embodiment of the protocol for synthesizing a stereospecific PS oligonucleotide
  • FIG. IB is a diagrammatic representation of another embodiment of the protocol for synthesizing a stereospecific PS oligonucleotide
  • FIG. 2A is an autoradiogram demonstrating the nuclease resistance of phosphodiester (PO) , stereospecific phosphorothioate (PS-Rp) , and synthetic phosphorothioate (PS) oligonucleotides in bovine serum;
  • PO phosphodiester
  • PS-Rp stereospecific phosphorothioate
  • PS synthetic phosphorothioate
  • FIG. 2B is an autoradiogram demonstrating the nuclease resistance of PO, PS-Rp, and PS oligonucleotides in human serum;
  • FIG. 3A is an autoradiogram demonstrating the nuclease resistance of PO, PS-Rp, and PS oligonucleotides in the presence of DNA polymerase I
  • FIG. 3B is an autoradiogram demonstrating the nuclease resistance of PO, PS-Rp, and PS oligonucleotides in the presence of T4 DNA polymerase;
  • FIG. 4A is a graphic representation of the hybridization affinity of PS-Rp and PS oligonucleotides to complementary DNA and RNA targets, as measured by melting temperature (T m ) ;
  • FIG. 4B is a graphic representation of the hybridization affinity of PS-Rp and PS oligonucleotides to complementary DNA and RNA targets, as measured by circular dichroism;
  • FIG. 5 is a graphic representation of the ability of PS-Rp ( ⁇ ) and PS oligonucleotides (D) to inhibit rHIV reverse transcriptase;
  • FIG. 6 is an autoradiogram demonstrating the susceptibility of PS-Rp and PS oligonucleotides to RNase H digestion.
  • the present invention provides for procedures for synthesizing stereospecific PS- oligonucleotides, and, in particular for preparing stereospecific (all Rp) oligonucleotide phosphorothioates using an enzymatic process. These Rp oligonucleotides appear to bind to proteins with less affinity and are believed to be less mitogenic than their non-stereospecific counterparts.
  • FIGS. 1A and IB The procedure established for synthesizing stereospecific PS-oligonucleotides (all Rp) is outlined in FIGS. 1A and IB. It has been shown that DNA polymerase uses only Sp diastereomers of 2' -deoxynucleotide 5'-( -thio) triphosphate during extension by single phosphorothioate to produce Rp linkages (Eckstein (1985) Ann. Rev. Biochem. 54: 367- 402) . However, such an extension was limited to incorporation of a single phosphorothioate linkage.
  • an appropriate template having the complementary nucleic acid sequence complementary to the desired sequence of the stereospecific molecule and a primer having a nucleic acid sequence complementary to a 3' portion of the template are designed.
  • Synthesis of the template is carried out using any method known in the art to produce phosphodiester-linker oligonucleotides.
  • One such useful method uses ⁇ - cyanoethyl phosphoramidite chemistry (Beaucage in Protocols for Oligonucleotides and Analogs , Methods in
  • RNA protocol such as desilylation
  • the primer is annealed to the template by methods well known in the art, such as by heating for 3 minutes at 90°C, and then cooling for one hour at room temperature.
  • the primer is then extended to form an oligonucleotide with a nucleic acid sequence complementary to the template.
  • Extension of the primer is carried out with any enzyme capable of such synthesis, such as Taq DNA polymerase, DNA polymerase I (Pol I polymerase) , T4 DNA polymerase, and reverse transcriptase using all four 2' -deoxynucleoside 5' - ( ⁇ -thio) triphosphates, or analogs of 2' -deoxynucleoside 5'-(o:-thio) triphosphates, as described by Eckstein ⁇ Ann. Rev.
  • any enzyme capable of such synthesis such as Taq DNA polymerase, DNA polymerase I (Pol I polymerase) , T4 DNA polymerase, and reverse transcriptase using all four 2' -deoxynucleoside 5' - ( ⁇ -thio) triphosphates, or analogs of 2' -deoxynucleoside 5'-(o:-thio) triphosphates, as described by Eckstein ⁇ Ann. Rev.
  • the newly synthesized stereospecific PS-Rp oligonucleotide is liberated by cleavage from the primer and release from the template. Liberation may be accomplished with any procedure that will cleave the PS-Rp oligonucleotide from its primer at the ribonucleotide and will cause the PS-Rp oligonucleotide to dissociate from its template.
  • the ribonucleotide may be at the 3' -penultimate (FIG. 1A) or 3'-terminal position (FIG. IB) of the primer before extension.
  • Useful treatments include cleavage with a strong alkaline which digests RNA or with an endoribonuclease or other enzyme which cleaves at an RNA/DNA junction.
  • Preferable alkalies include halide hydroxides such as sodium hydroxide, lithium hydroxide, ammonium hydroxide, and potassium hydroxide, with the most preferable being ammonium hydroxide or potassium hydroxide.
  • Useful enzymes include RNase A, RNase Tl, RNase T2, RNase U2, RNase Nl, and RNase N2, which are commercially available.
  • the product is purified by chromatography, gel electrophoresis, ultracentrifugation, or any other method known in the art that separates single-stranded oligonucleotides from themselves and from double- stranded molecules.
  • PAGE for example, on a 20% polyacrylamide gel containing 8 M urea is useful for this purpose.
  • the band containing the PS-Rp oligonucleotide is purified by locating it, for example, by UV shadowing, and excising and desalted it. Generally, if synthesis is carried out using about 8 nmoles of primer and 2.5 nmoles of template, about 1 nmole of PS-Rp oligonucleotide product is obtained.
  • the oligonucleotides of the invention are useful as modulators of gene expression.
  • an oligonucleotide of the invention can be targeted to that gene in a tissue culture system and the effects of this modulation studied.
  • the oligonucleotides of the invention can be used as a tool to suppress the production of the protein by targeting the nucleic acid that encodes the protein and then observing the biological effects brought about the suppression of the protein. In such a situation, it is not the integrated oligonucleotide that is being studied but the effects of suppressing production of the protein.
  • Use of the oligonucleotides of the invention in this way provides an easily executed alternative to the laborious method of mutating the targeted gene, and thereby creating a deletion mutant.
  • the PS-Rp oligonucleotides of the invention are useful as stereochemical probes of phospho- and nucleotidyltranserase, as these oligonucleotides can distinguish between the incorporation of a deoxyribonucleotide into a growing polymer via a single bond cleavage at the phosphorus of the nucleotide triphosphate as the 3'-hydroxyl of the growing chain displaces the pyrophosphate, or via two bond cleavages if a covalent nucleotidyl enzyme is involved (reviewed in Brody et al . (1981) Biochem.20 :1245-1252) .
  • the comparable stability of a PS-Rp oligonucleotide prepared according to the method of the invention was determined by exposing it, as well as sequence-comparable PO- and PS-linked oligonucleotides, to a nuclease source such as human or bovine serum.
  • a nuclease source such as human or bovine serum.
  • FIG. 2A show that PS-Rp oligonucleotides are still present after 6 hours and up to 24 hours incubation in bovine serum, whereas PO oligonucleotides are not.
  • human serum FIG. 2B
  • PS-Rp and PS oligonucleotides are still present after 120 minutes of incubation, whereas PO-linked oligonucleotides were digested after 60 minutes of incubation.
  • PS-Rp oligonucleotides are more stable than phosphodiester-linked oligonucleotides, and have stability comparable to PS oligonucleotides in bovine and human serum, and in the presence of DNA polymerase and T 4 DNA polymerase.
  • PS-Rp oligonucleotide and synthetic PS- oligonucleotide were also compared for their affinity for complementary DNA and RNA targets, as measured by melting temperature( T m ) and Circular dichroism. As shown in FIG. 4A, both PS-Rp and PS-oligonucleotides showed the same T m with DNA target (51.8° and 51.9°) .
  • PS-Rp oligonucleotide had a higher T m (68.9°) compared to synthetic PS-oligonucleotide (64°) with an RNA target, indicating that it has a greater binding affinity for its target.
  • FIG. 4B demonstrates that the difference in CD spectra for RNA duplexes is more significant than that for DNA duplexes, indicating that RNA target is more sensitive to the conformational change of PS-Rp versus PS oligonucleotides at hybridization than the DNA target.
  • PS oligonucleotides present as mixture of Rp and Sp diastereomers are to inhibit the enzymatic activity of reverse transcriptase.
  • PS-Rp oligonucleotides and PS oligonucleotides were incubated individually with reverse transcriptase. As shown in FIG. 5, the PS-Rp oligonucleotide was found to be a stronger inhibitor of reverse transcriptase than the synthetic PS-oligonucleotide mixture.
  • PS-Rp oligonucleotides were also subjected to RNase H digestion to determine if their ability to trigger this degradative response is comparable to that of the PS diastereomeric mixtures.
  • FIG. 6 shows that PS-Rp oligonucleotides are better substrates for RNase H than the synthetic PS-oligonucleotide mixture.
  • stereospecific PS-Rp oligonucleotides have similar if not improved antisense characteristics as a diastereomeric Rp + Sp mixture of PS oligonucleotides.
  • the reaction was initiated by the addition of 2500 units of sequence grade Taq DNA polymerase (Promega Corp., Madison, I) . The mixture was incubated for 4 hours at 37°C. The reaction mixture was desalted and treated with of ammonium hydroxide solution (28-30%) at 55°C for 24 hours, or with 0.3 M KOH at 37°C for 1 hour. In other tests, the desalted reaction mixture is alternatively treated with RNase A , RNase Tl,
  • RNase T2, RNase U2, RNase Nl, or RNase N2 which are available from the Sigma Chemical Co. (St. Louis, MO) at 95°C for about 3 minutes.
  • the product was dried and purified by PAGE on a 20% denaturing polyacrylamide gel.
  • the product was excised from the gel under UV shadowing and eluted with 500 mM ammonium acetate and desalted by dialysis, as described in FIG. 1.
  • PS-oligonucleotide The stability of phosphodiester-linked (PO) , stereoregular (PS-Rp) , and synthetic PS- oligonucleotide (PS) in bovine and human serum was tested as follows. 60 pmole of [ 32 P] 5' -end- labelled oligonucleotide was incubated in 100 ul of human serum (Sigma Chemical Co., St. Louis, MO) at 37°C. Aliquots of 20 ⁇ l were sampled at time 0, 30, 60, and 120 minutes, and then incubated with 20 ⁇ l of 20 mM Tris, pH 7.8, 10 mM NaCl, 10 mM EDTA, 0.5% SDS, and 100 ⁇ g proteinase K at 65°C for one hour.
  • human serum Sigma Chemical Co., St. Louis, MO
  • the samples were extracted by phenol/chloroform, ethanol precipitated, and then analyzed by PAGE in gels containing 20% acrylamide, 8.3 M urea.
  • the gel was fixed in a acetic acid/methanol/water (10:10:80) , v/v/v) solution and dried for 3 hours before being subjected to autoradiography.
  • the autoradiograms are shown in FIGS. 2A (bovine serum) and 2B (human serum) .
  • DNA polymerase I DNA polymerase
  • the DNA polymerase I assay was carried out in 20 ⁇ l volume containing 30 pmoles [ 32 P] -5' end labelled PO, PS, or PS-Rp oligonucleotide, 50 nm Tris, pH 8.0, 5 mM MgCl 2 , 5 mM DTT, 0.05% bovine serum albumin, and 5 units of DNA polymerase I (Worthington Biochemical Corp., Freehold, NJ) . An aliquot was removed at time 0 and the remaining was incubated at 37°C. 5 ⁇ l aliquots of were removed at times 1, 2, and 4 hours.
  • the T4 DNA polymerase assays were carried out in 30 ⁇ l volume containing 45 pmoles [ 32 P]-5' end labelled PO, PS, or PS-Rp oligonucleotide, 50 mM Tris, pH 8.0, 5 mM MgCl 2 , 5 mM DTT, 0.05% bovine serum albumin, and 7.5 units of T4 DNA polymerase (Promega, Madison, WI) . An aliquot was removed at time 0 and the remaining was incubated at 37°C. 5 ⁇ l aliquots were removed at times 5, 15, 30, and 60 minutes.
  • T m Melting temperature studies were carried as follows. 1.06 x 10 "6 M PS-Rp or PS oligonucleotides were placed in 10 mM phosphate, 100 mM NaCl, pH 7, subjected to temperatures ranging from 30 to 75°C, and the absorbance at A 260 measured using a spectrophotometer (GBC 920, GBC, Victoria, Australia) . The results are shown in FIG. 4A.
  • Circular dichroism studies were performed to determine the relative binding affinity of PS-Rp and PS oligonucleotides to complementary RNA or DNA sequences. 1.06 x 10 "6 M PS-Rp or PS oligonucleotides were placed in 10 mM phosphate, 10 mM NaCl, pH 7.0, and subjected to 200-320 nm UV light using a spectropolarimeter (J-710, Jasco, Easton, MD) . The results are shown in FIG. 4B.
  • dNTPs deoxynucleoside 5' -triphosphates
  • Activity of the PS-Rp oligonucleotides is measured by assaying their ability to inhibit HIV- 1 expression in H9, MOLT-3, or other cells.
  • 5 x 10 s cells per ml are infected with 2.5-5 x 10 8 virus particles of HIV-1 strains HTLV- IIIB or HTLV-IIIC.
  • Infection with 500-1000 virus particles per cell represents a multiplicity of infection (MOD of 0.5-1.
  • Infection of cells is carried out by simultaneous addition of virus and antisense oligomers to the cells cultured in medium containing RPMI 1640 (10% (vol/vol) fetal bovine serum, 2 mM glutamine, and 250 ⁇ l of gentamicin per ml) in a humidified atmosphere containing 5% C0 2 at 37°C. After 4 days, the cells and supernatant are examined for the level of HIV-1 expression by measuring syncytia (MOLT-3 cells) and viral antigen expression as well as cell viability. The number of syncytia formed in MOLT-3 cells is counted after triturating the cells to obtain an even distribution of the syncytia in the culture.
  • RPMI 1640 % (vol/vol) fetal bovine serum, 2 mM glutamine, and 250 ⁇ l of gentamicin per ml
  • the average number of syncytia is obtained by counting several fields in duplicate cultures. Cell viability is measured in the presence of trypan blue, and the cells that excluded the dye are counted as viable cells. HIV-1 antigen expression is measured in cells fixed in methanol/acetone. Briefly, the cells were pelleted and then resuspended in phosphate- buffered saline (PBS) at a concentration of 10 6 -20- cells per ml. The cells are then spotted on toxoplasmosis slides, air-dried, and fixed in methanol/acetone (1:1, vol/vol) for 15 min at room temperature. The slides are next incubated with 10% normal goat serum at room temperature for 30 min and washed with PBS (four times) .
  • PBS phosphate- buffered saline
  • HIV-1 p24 or pl7 monoclonal antibody are added to each well and the slides incubated for 30 min in a humid chamber at 37°C.
  • the slides are washed with PBS (four times) , incubated with fluorescein isothiocyanate-labeled goat anti-mouse IgG (Cappel Laboratories, Cochranville, PA) for 30 min at 37°C, and then washed with PBS overnight.
  • the slides are counterstained with Evan's blue, washed with PBS, mounted with 50% glycerol, and examined with a Zeiss fluorescence microscope. The percentages of cells fluorescing in the oligomer- treated and untreated cultures are compared.
  • the cells are lysed for 10 min at 4°C in 450 ⁇ l of buffer A (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.5 mM MgCl 2 ) and 50 ⁇ l of 5% Nonidet P-40 (NP-40) . After centrifugation at 2000 rpm for 5 min, the upper cytoplasmic fraction (combined cytosol and cell membrane) is removed and the pellet (nuclei) dissolved in 500 ⁇ l of buffer A and measured for radioactivity.
  • buffer A 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.5 mM MgCl 2
  • NP-40 Nonidet P-40
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE CDNA/RNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE RNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES

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Abstract

L'invention concerne un procédé de synthèse d'oligonucléotides de phosphorothioates stéréospécifiques (Rp). Ce procédé consiste à hybrider à une matrice une amorce composée d'une pluralité de désoxyribonucléotides et d'un ribonucléotide au niveau de la position terminale 5' ou de la position pénultième 5'. Cette structure est mise en contact avec un mélange de diastéréomères Sp de α-triphosphate de désoxynucléosides et d'une ADN polymérase afin qu'une extension d'oligodeoxynucléotides PS-Rp soit formée et libérée en tant qu'oligonucléotide monocaténaire PS-Rp par coupure après le ribonucléotide de l'amorce. L'invention concerne également des oligonucléotides PS-Rp, ainsi que des oligonucléotides préparés selon ce procédé.
EP95943748A 1994-12-22 1995-12-12 Synthese d'oligonucleotides de phosphorothioates stereospecifiques Pending EP0807171A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US362631 1989-06-07
US36263194A 1994-12-22 1994-12-22
PCT/US1995/016086 WO1996019572A1 (fr) 1994-12-22 1995-12-12 Synthese d'oligonucleotides de phosphorothioates stereospecifiques

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EP0807171A1 true EP0807171A1 (fr) 1997-11-19

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EP (1) EP0807171A1 (fr)
JP (1) JPH10511267A (fr)
CN (1) CN1175281A (fr)
AU (1) AU4514696A (fr)
CA (1) CA2208528A1 (fr)
WO (1) WO1996019572A1 (fr)

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WO1996019572A1 (fr) 1996-06-27
CA2208528A1 (fr) 1996-06-27

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