EP0476071A1 - Exonuclease-resistant oligonucleotides and methods for preparing the same - Google Patents
Exonuclease-resistant oligonucleotides and methods for preparing the sameInfo
- Publication number
- EP0476071A1 EP0476071A1 EP90913804A EP90913804A EP0476071A1 EP 0476071 A1 EP0476071 A1 EP 0476071A1 EP 90913804 A EP90913804 A EP 90913804A EP 90913804 A EP90913804 A EP 90913804A EP 0476071 A1 EP0476071 A1 EP 0476071A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- oligonucleotide
- linkages
- hydrogen
- phosphoramidate
- phosphorothioate
- 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.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Definitions
- phosphodiester linkages are replaced with a specified number of phosphoramidate linkages.
- It a further object of the invention to provide methods of making such exonuclease-resistant oligonucleotides. It is still a further object of the invention to provide a method for end-capping oligonucleotides with moieties which can perform multiple functions, such as aiding in transport, serving as chromophoric tags, or enabling cross-linking.
- the present invention provides oligonucleotides having two or more phosphoramidate linkages at the 3' terminus and/or 5' terminus, which oligonucleotides are resistant to
- the number of phosphoramidate linkages is at least 1 and less than a number which would interfere with hybridization to a complementary oligonucleotide strand, and/or less than a number which would interfere with RNAse activity when said oligonucleotide is hybridized to RNA.
- phosphoramidate linkages are incorporated at either or both the 3' terminus and the 5' terminus.
- the phosphoramidate linkages may be substituted with any one of a number of different types of moieties as will be described in detail hereinbelow.
- exonuclease-resistant are provided which have the following formulas I, II or III, i.e., containing phosphoramidate linkages as just described as well as phosphoromonothioate and/or
- each n, m, i, j and s is independently an integer and each s is in the range of about 2 to 10; each n and m is independently from 1 to about 50; s + n in formulas I and II is less than 100; and s + s + m in formula III is less than about 100; each i varies from 1 to n; each j varies from 1 to m; T is hydrogen or a hydroxyl- protecting group; R 1 and R 2 are moities independently selected from the group consisting of hydrogen,
- each B is independently a protected or unprotected heterocyclic base
- each X i and X j is independently O or S ; and each Y i and Y j is independently R, -SR or -OR, where R is as defined for R 1 and R 2 .
- the present invention also provides methods for preparing such end-capped oligonucleotides.
- pyrimidine base or modified purine or pyrimidine base.
- polynucleotide and oligonucleotide
- nucleoside and “nucleotides” will include those moieties which contain not only the known purine and pyrimidine bases, i.e., adenine, thymine, cytosine, guanine and uracil, but also other heterocyclic bases which contain protecting groups or have been otherwise modified or derivatized.
- 5-chlorouracil 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
- 5-carboxymethylaminomethyl-2-thiouridine 5-carboxy- methylaminomethyluracil, dihydroura ⁇ il, beta-D-galacto- sylqueosine, inosine, N6-isopentenyladenine, 1-methyl- adenine, 1-methylpseudouracil, 1-methylguanine,
- Modified nucleosides or nucleotides can also include modifications on the sugar moiety, for example, wherein one or more of the hydroxyl groups are replaced with halogen or aliphatic groups, or functionalized as ethers, amines, etc.
- Internucleotide phosphodiester linkages are prepared from hydrogen phosphonate linkages preferably by oxidation with, e.g., aqueous iodine.
- the oligonucleotide In order that the oligonucleotide be resistant to such enzymatic degradation, it is modified so that phosphodiester linkages initially present at the 3' terminus are replaced with a selected number of phosphoramidate linkages, that number being at least one and less than a number which would cause interference with hybridization to a complementary oligonucleotide strand, and/or less than a number which would interfere with RNAseH activity when said the oligonucleotide is
- the temperature of a duplex formed is lowered by less than about 5°C.
- the number of phosphoramidate linkages present is typically and preferably between about 2 and 10, more preferably between about 2 and 8, and most preferably between about 2 and 6.
- the phosphoramidate linkage has the formula
- R 1 and R 2 are not both hydrogen, i.e., the phosphoramidate linkages herein are always N-substituted. In this case, it is preferred that one of the two substituents be hydrogen.
- Suitable hydrocarbyl and oxyhydrocarbyl substituents include, for example, linear or branched alkyl of 1-20 carbon atoms, linear or branched alkenyl of 2-20 carbon atoms, cycloalkyl or cycloalkenyl of 3-20 carbon atoms, linear or branched alkoxy of 1-20 carbon atoms, or aryl of 6-18 carbon atoms.
- Examples of preferred oligonucleotide linkages within the aforementioned groups are wherein one of R 1 and R 2 is H and the other is either 2-methoxyethyl, dodecyl, or n- propyl.
- the 2-methoxyethyl and dodecyl linkages are sometimes referred to herein as "MEA” and "C12",
- the R 1 and R 2 groups may also be, in addition to the foregoing, macromolecular species such as sugars, polypeptides, chromophoric groups, lipophilic groups, polymers, steroid hormones, or the like.
- Lipophilic groups refer to moieties which are chemically compatible with the outer cell surface, i.e., so as to enable the oligonucleotide to attach to, merge with and cross the cell membrane. Examples of such lipophilic groups are fatty acids and fatty alcohols (in addition to the long chain hydrocarbyl groups described above).
- R 1 and/or R 2 examples include transferrin and epidermal growth factor (EGF), while suitable non-polypeptide polymers include ionic, nonionic and zwitterionic
- polymers examples of a particularly preferred polymer is polyethylene glycol.
- Steroid substituents include any of the general fat ⁇ ily of lipid compounds which comprise sterols,
- bioacids cardiac glycosides, seponans, and sex hormones, which include the following basic structure:
- steroids examples include natural corticosteroid hormones (produced by the adrenal glands), sex hormones (progesterone, androgens, and estrogens).
- R 1 and R 2 groups can confer any of a variety of desired properties to the oligo- nucleotide.
- R 1 or R 2 is a polymer such as polyethylene glycol, a polypeptide or a lipophilic group such as a long-chain hydrocarbyl moiety, such a group may facilitate transport or permeation of the oligonucleotide through cell membranes, thus increasing the cellular uptake of the oligonucleotide.
- oligonucleotides of the present invention can include other phosphoramidate N-substituents not explicitly disclosed herein so long as those substituents confer exonuclease resistance and do not interfere with hybridization to a complementary oligonucleotide strand.
- the invention also encompasses oligonucleotide compositions containing oligonucleotides of the following formula I, II or III, i.e., wherein phosphoromonothioate and/or phosphorodithioate linkages are incorporated in addition to the phosphoramidate linkages:
- the 3'-capped oligonucleotides may be prepared by first preparing a polymer-bound polynucleoside with the formula IV
- P is a solid state polymeric support, or other type of solid support
- B the base portion of a nucleoside, i.e., a purine or pyrimidine base, or any modified purine or pyrimidine base.
- the functional groups on the base i.e., the amine groups, will be appropriately protected during the course of the synthesis and removed after the completed polynucleotide is removed from the polymer support.
- the linkage to the polymer support is through the 3' hydroxy group, the free hydroxy group is the 5' group of the nucleoside.
- the group T is a
- the polymer-bound polynucleoside hydrogen phosphonate (IV) is preferably prepared by treating the DBU
- the oligonucleotide chain elongation will proceed in conformance with a predetermined sequence in a series of condensations, each one of which results in the addition of another nucleoside to the oligomer.
- the condensation is typically accomplished with dehydrating agents, which are suitably phosphorylating agents or acylating agents such as isobutylchloroformate,
- diphenylchlorophosphate organic acid anhydrides (such as acetic anhydride, isobutyric anhydride or trimethyl acetic anhydride) and organic acid halides such as pivaloyl chloride, pivaloyl bromide, 1-adamantyl- carboxylic chloride or benzoyl chloride.
- organic acid anhydrides such as acetic anhydride, isobutyric anhydride or trimethyl acetic anhydride
- organic acid halides such as pivaloyl chloride, pivaloyl bromide, 1-adamantyl- carboxylic chloride or benzoyl chloride.
- the preferred condensing agent is pivaloyl chloride in pyridine
- the oligonucleotide is then completed by methods which form nonphosphoramidate linkages, such as phosphodiester linkages,
- the preferred method for completing the oligonucleotide is to continue the sequence using 5'-protected nucleoside hydrogen- phosphonates. In the instance where the 5' end will not be capped, after the last 5'-protected nucleoside
- the oligonucleotide may be separated from the carrier, using conventional methods, which in the preferred instance is incubation with concentrated ammonium hydroxide. Any protecting groups may be removed as described above using about 2% dichloroacetic acid/CH 2 Cl 2 , or about 80% acetic acid, or by other conventional methods, depending on the nature of the protecting groups. The desired oligonucleotide is then purified by HPLC, polyacrylamide gel electro- phoresis or using other conventional techniques.
- i varies from 1 to 5
- B a purine or pyrimidine base
- a combination of both of the above methods for making a 5' and a 3' end- capped oligonucleotide may be utilized.
- the first two (or more) internucleotide linkages on the 3'-bound oligonucleotide may be oxidized to form the
- the non-terminal portion of the oligonucleotide may be made (having
- 5'- or 3'-phosphoramidate-capped oligonucleotides as made in accordance with the present invention may be as therapeutic agents against viral diseases (such as HIV, hepatitis B, cytomegalovirus), cancers (such as leukemias, lung cancer, breast cancer, colon cancer) or metabolic disorders, immune modulation agents, or the like, since the present end-capped oligonucleotides are stable within the environment of a cell as well as in extracellular fluids such as serum, and can be used to selectively block protein synthesis, transcription, replication of RNA and/or DNA which is uniquely associated with the disease or disorder.
- viral diseases such as HIV, hepatitis B, cytomegalovirus
- cancers such as leukemias, lung cancer, breast cancer, colon cancer
- immune modulation agents or the like
- the end-capped oligonucleotides of the invention may also be used as therapeutics in animal health care, plant gene regulation (such as plant growth promoters) or in human diagnostics, such as to stabilize DNA probes to detect microorganisms, oncogenes, genetic defects, and the like, and as research reagents to study gene functions in animal cells, plant cells, microorganisms, and viruses.
- plant gene regulation such as plant growth promoters
- human diagnostics such as to stabilize DNA probes to detect microorganisms, oncogenes, genetic defects, and the like
- research reagents to study gene functions in animal cells, plant cells, microorganisms, and viruses.
- dermatologic applications for treatment of diseases or for cosmetic purposes.
- the oligomer was removed from the solid support, deprotected with cone. NH 4 OH (45°C/18 hr.), and purified by HPLC (PRP) using an acetonitrile (CH 3 CN) gradient in 50 mM aqueous TEAP.
- the DMT was removed from the product fraction (80% acetic acid/R.T./2 hrs.), evaporated, desalted and evaporated. Approximately 1 ⁇ g of purified product was 5' end-labeled with T4 poly- nucleotide kmase and ⁇ - 32 P ATP for further
- Polymer-bound polynucleoside H-phosphonates were prepared as in the preceding example on control pore glass using the DBU salt of the protected nucleoside H-phosphonate. After twelve couplings the polynucleoside H-phosphonate was oxidized with aq. I 2 (0.1 M in N-methyl morpholine/water/THF, 5:5:90) followed by two more couplings and oxidation with a solution of 2-methoxy- ethylamine in Pyr/CCl 4 (1:5:5) (20 min.) to generate a 15-mer containing twelve diester linkages at the 3' end and two phosphoramidate linkages at the 5' end.
- aq. I 2 0.1 M in N-methyl morpholine/water/THF, 5:5:90
- 2-methoxy- ethylamine in Pyr/CCl 4 (1:5:5) 20 min.
- Example 2 The procedure of Example 2 was repeated using propylamine in place of 2-methoxyethylamine, so as to yield a 15-mer containing twelve diester linkages at the 3' end and two phosphoramidate linkages at the 5' end, wherein the phosphoramidate linkages are substituted as in the preceding example, i.e., one of R 1 and R 2 is hydrogen and the other is n-propyl.
- Example describes hybridization stability studies performed using end-capped oligonucleotides as described and claimed herein.
- Oligonucleotides containing end-caps were tested for their ability to form stable duplexes with complementary single-stranded DNA sequences; the various oligonucleotides tested were outlined below in Table 1. Duplex stability was measured by determining the melting temperature T m in solution over a range of temperatures. The experiment was conducted in a solution containing 150 mM NaCl, 5 mM Na 2 HPO 4 and 3 ⁇ M DNA at a pH of 7.1. The results obtained and set forth in Table 1 show that binding to complementary sequences is not materially affected by 3'-end-cap modification.
- the acute infection assay used the MOLT-4 cell line which is susceptible to HIV infection. Measurement of HIV p24 was used to assay for inhibition of virus replication 7 days after infection with virus at a multiplicity of infection of approximately 0.1.
- oligonucleotide Approximately 1 x 106 cells were preincubated with oligonucleotide, washed, infected with virus stock and then incubated for 7 days in oligonucleotide. HIV p24 levels in the supernatant were measured by
- Toxieity data was obtained by incubation of 3'-end-capped oligonucleotides with uninfected cells, followed by a comparison with cell numbers with control cultures incubated in the absence of oligonucleotide. Toxieity results are expressed as the percent reduction of cell numbers obtained by incubation in oligonucleotide for 7 days compared to controls.
- the effective inhibition of HIV replication using low levels (0.5 to 5 ⁇ M) of capped oligodeoxynucleotides supports the conclusion that significant nuclease degradation of the oligonucleotides of the invention does not occur either extracellularly or intracellulary.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
On prévoit un procédé pour la préparation d'oligonicleotides ayant des terminaisons 3' et/ou 5' de manière à rendre l'oligonucléotide résistant à la décomposition provoquée par des exonucléasés. Ladite résistance est réalisée en incorporant deux ou plusieurs liaisons de phosphoramidate et phosphoromonothioate et/ou phosphorodithioate aux terminaisons 5' et/ou 3' de l'olignucléotide, où le nombre de liaisons de phosphoramidate est inférieur à un nombre pouvant perturber l'hybridation avec un brin d'oligonucléotide complémentaire et/ou pouvant perturber une activité ARNseH lorsque l'oligonucléotide est hybridé avec l'ARN.A process is provided for the preparation of oligonicleotides having 3 'and / or 5' terminations so as to render the oligonucleotide resistant to decomposition caused by exonucleones. Said resistance is achieved by incorporating two or more phosphoramidate and phosphoromonothioate and / or phosphorodithioate bonds at the 5 ′ and / or 3 ′ ends of the olignucleotide, where the number of phosphoramidate bonds is less than a number which can disturb hybridization with a strand of oligonucleotide complementary and / or capable of disturbing an RNAseH activity when the oligonucleotide is hybridized with RNA.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US36104589A | 1989-06-05 | 1989-06-05 | |
US361045 | 1989-06-05 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0476071A1 true EP0476071A1 (en) | 1992-03-25 |
EP0476071A4 EP0476071A4 (en) | 1992-11-04 |
Family
ID=23420432
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900913804 Withdrawn EP0476071A4 (en) | 1989-06-05 | 1990-06-05 | Exonuclease-resistant oligonucleotides and methods for preparing the same |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0476071A4 (en) |
JP (1) | JPH05500799A (en) |
KR (1) | KR920701230A (en) |
CA (1) | CA2058632C (en) |
WO (1) | WO1990015065A1 (en) |
Families Citing this family (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5149797A (en) * | 1990-02-15 | 1992-09-22 | The Worcester Foundation For Experimental Biology | Method of site-specific alteration of rna and production of encoded polypeptides |
US5220007A (en) * | 1990-02-15 | 1993-06-15 | The Worcester Foundation For Experimental Biology | Method of site-specific alteration of RNA and production of encoded polypeptides |
US5965722A (en) * | 1991-05-21 | 1999-10-12 | Isis Pharmaceuticals, Inc. | Antisense inhibition of ras gene with chimeric and alternating oligonucleotides |
WO1994008003A1 (en) * | 1991-06-14 | 1994-04-14 | Isis Pharmaceuticals, Inc. | ANTISENSE OLIGONUCLEOTIDE INHIBITION OF THE ras GENE |
AU3250093A (en) | 1991-12-12 | 1993-07-19 | Gilead Sciences, Inc. | Nuclease stable and binding competent oligomers and methods for their use |
US5792608A (en) * | 1991-12-12 | 1998-08-11 | Gilead Sciences, Inc. | Nuclease stable and binding competent oligomers and methods for their use |
US5434257A (en) * | 1992-06-01 | 1995-07-18 | Gilead Sciences, Inc. | Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages |
ATE162198T1 (en) * | 1992-07-27 | 1998-01-15 | Hybridon Inc | OLIGONUCLEOTIDE ALKYLPHOSPHONOTHIATE |
US5580972A (en) * | 1993-06-14 | 1996-12-03 | Nexstar Pharmaceuticals, Inc. | Purine nucleoside modifications by palladium catalyzed methods |
US5719273A (en) * | 1993-06-14 | 1998-02-17 | Nexstar Pharmaceuticals, Inc. | Palladium catalyzed nucleoside modifications methods using nucleophiles and carbon monoxide |
CA2185239C (en) * | 1994-03-16 | 2002-12-17 | Nanibhushan Dattagupta | Isothermal strand displacement nucleic acid amplification |
EP0830368A1 (en) | 1995-06-07 | 1998-03-25 | Genta Incorporated | Novel carbamate-based cationic lipids |
US6111095A (en) * | 1995-06-07 | 2000-08-29 | Merck & Co., Inc. | Capped synthetic RNA, analogs, and aptamers |
AU2542097A (en) * | 1996-03-26 | 1997-10-17 | Lynx Therapeutics, Inc. | Oligonucleotide treatments and compositions for human melanoma |
US5959100A (en) * | 1996-03-27 | 1999-09-28 | Nexstar Pharmaceuticals, Inc. | Pyrimidine nucleosides as therapeutic and diagnostic agents |
US5945527A (en) * | 1996-05-30 | 1999-08-31 | Nexstar Pharmaceuticals, Inc. | Palladium catalyzed nucleoside modification methods using nucleophiles and carbon monoxide |
US6576752B1 (en) | 1997-02-14 | 2003-06-10 | Isis Pharmaceuticals, Inc. | Aminooxy functionalized oligomers |
US6172209B1 (en) | 1997-02-14 | 2001-01-09 | Isis Pharmaceuticals Inc. | Aminooxy-modified oligonucleotides and methods for making same |
US6127533A (en) * | 1997-02-14 | 2000-10-03 | Isis Pharmaceuticals, Inc. | 2'-O-aminooxy-modified oligonucleotides |
US6028183A (en) | 1997-11-07 | 2000-02-22 | Gilead Sciences, Inc. | Pyrimidine derivatives and oligonucleotides containing same |
US6007992A (en) * | 1997-11-10 | 1999-12-28 | Gilead Sciences, Inc. | Pyrimidine derivatives for labeled binding partners |
US6673912B1 (en) | 1998-08-07 | 2004-01-06 | Isis Pharmaceuticals, Inc. | 2′-O-aminoethyloxyethyl-modified oligonucleotides |
US6043352A (en) | 1998-08-07 | 2000-03-28 | Isis Pharmaceuticals, Inc. | 2'-O-Dimethylaminoethyloxyethyl-modified oligonucleotides |
US6020483A (en) | 1998-09-25 | 2000-02-01 | Nexstar Pharmaceuticals, Inc. | Nucleoside modifications by palladium catalyzed methods |
US6207819B1 (en) * | 1999-02-12 | 2001-03-27 | Isis Pharmaceuticals, Inc. | Compounds, processes and intermediates for synthesis of mixed backbone oligomeric compounds |
AU2001262108A1 (en) * | 2000-03-17 | 2001-09-24 | Unilever Plc | Non-physiologic dna fragments for tanning skin |
EP1322656B1 (en) * | 2000-09-26 | 2008-01-16 | Idera Pharmaceuticals, Inc. | Modulation of immunostimulatory activity of immunostimulatory oligonucleotide analogs by positional chemical changes |
CN101671335A (en) | 2001-05-31 | 2010-03-17 | 梅达莱克斯公司 | Cytotoxins, prodrugs, linkers and stabilizers useful therefor |
US7255874B1 (en) | 2001-12-21 | 2007-08-14 | Closure Medical Corporation | Biocompatible polymers and adhesives: compositions, methods of making and uses related thereto |
US7125945B2 (en) | 2003-09-19 | 2006-10-24 | Varian, Inc. | Functionalized polymer for oligonucleotide purification |
NZ566982A (en) | 2005-09-26 | 2011-06-30 | Medarex Inc | Duocarmycin drug conjugates |
US7947447B2 (en) | 2007-01-16 | 2011-05-24 | Somalogic, Inc. | Method for generating aptamers with improved off-rates |
EP3216874A1 (en) | 2008-09-05 | 2017-09-13 | TOMA Biosciences, Inc. | Methods for stratifying and annotating cancer drug treatment options |
MX2020003168A (en) * | 2010-04-12 | 2022-05-31 | Somalogic Inc | 5-position modified pyrimidines and their use. |
AU2014353102B2 (en) | 2013-11-21 | 2019-05-16 | Somalogic Operating Co., Inc. | Cytidine-5-carboxamide modified nucleotide compositions and methods related thereto |
US20160017394A1 (en) * | 2014-07-15 | 2016-01-21 | Life Technologies Corporation | Compositions and methods for nucleic acid assembly |
KR20230130175A (en) | 2014-12-26 | 2023-09-11 | 에모리 유니버시티 | N4-hydroxycytidine and derivatives and anti-viral uses related thereto |
AU2018378832B9 (en) | 2017-12-07 | 2021-05-27 | Emory University | N4-hydroxycytidine and derivatives and anti-viral uses related thereto |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0061746A1 (en) * | 1981-03-27 | 1982-10-06 | University Patents, Inc. | Phosphoramidite compounds and their use in producing oligonucleotides |
WO1986007362A1 (en) * | 1985-06-14 | 1986-12-18 | University Patents, Inc. | Method for synthesizing deoxyoligonucleotides |
WO1988000201A1 (en) * | 1986-06-24 | 1988-01-14 | California Institute Of Technology, Inc. | Novel deoxyribonucleoside phosphoramidites and their use for the preparation of oligonucleotides |
WO1989005358A1 (en) * | 1987-11-30 | 1989-06-15 | University Of Iowa Research Foundation | Dna and rna molecules stabilized by modifications of the 3'-terminal phosphodiester linkage and their use as nucleic acid probes and as therapeutic agents to block the expression of specifically targeted genes |
-
1990
- 1990-06-05 KR KR1019910701767A patent/KR920701230A/en not_active Application Discontinuation
- 1990-06-05 WO PCT/US1990/003138 patent/WO1990015065A1/en not_active Application Discontinuation
- 1990-06-05 CA CA002058632A patent/CA2058632C/en not_active Expired - Lifetime
- 1990-06-05 JP JP2509531A patent/JPH05500799A/en active Pending
- 1990-06-05 EP EP19900913804 patent/EP0476071A4/en not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0061746A1 (en) * | 1981-03-27 | 1982-10-06 | University Patents, Inc. | Phosphoramidite compounds and their use in producing oligonucleotides |
WO1986007362A1 (en) * | 1985-06-14 | 1986-12-18 | University Patents, Inc. | Method for synthesizing deoxyoligonucleotides |
WO1988000201A1 (en) * | 1986-06-24 | 1988-01-14 | California Institute Of Technology, Inc. | Novel deoxyribonucleoside phosphoramidites and their use for the preparation of oligonucleotides |
WO1989005358A1 (en) * | 1987-11-30 | 1989-06-15 | University Of Iowa Research Foundation | Dna and rna molecules stabilized by modifications of the 3'-terminal phosphodiester linkage and their use as nucleic acid probes and as therapeutic agents to block the expression of specifically targeted genes |
Non-Patent Citations (1)
Title |
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See also references of WO9015065A1 * |
Also Published As
Publication number | Publication date |
---|---|
CA2058632C (en) | 2004-08-24 |
EP0476071A4 (en) | 1992-11-04 |
WO1990015065A1 (en) | 1990-12-13 |
KR920701230A (en) | 1992-08-11 |
CA2058632A1 (en) | 1990-12-06 |
JPH05500799A (en) | 1993-02-18 |
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