WO1989009780A1 - Oligodesoxynucleotides marques au tritium specifiques a un site - Google Patents

Oligodesoxynucleotides marques au tritium specifiques a un site Download PDF

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Publication number
WO1989009780A1
WO1989009780A1 PCT/US1989/001535 US8901535W WO8909780A1 WO 1989009780 A1 WO1989009780 A1 WO 1989009780A1 US 8901535 W US8901535 W US 8901535W WO 8909780 A1 WO8909780 A1 WO 8909780A1
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labeled
oligonucleotide
tritium
nucleotide
product
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PCT/US1989/001535
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English (en)
Inventor
Frank Charles Richardson
Thomas Richard Skopek
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Chemical Industry Institute Of Toxicology
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Publication of WO1989009780A1 publication Critical patent/WO1989009780A1/fr

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    • 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

  • This invention relates to the formation of new nucleotides and oligonudeotides.
  • this invention relates to the formation of protected tritium- labeled nucleotide phosphoramidites and oligonudeotides with tritium located at predetermined nucleotide sites.
  • Radioactively tagged nucleotides Numerous investigators have incorporated radioactive labels into the nucleotides and other cell components of growing cells by growing the cells in media containing isotopically labeled compounds. See, for example, Oldham (U.S. Patent No. 3,854,240). The disclosure of this reference and of all other cited publications and patents is incorporated in full by reference herein. Most work using radioactively labeled nucleotides has involved use of nonspecific nucleotide labels such as in a uniformly labeled oligonucleotide to determine the presence of the particular oligonucleotide in organisms, tissues or cells. Thus, Stabinsky (U.S. Patent No.
  • the oligonucleotide contains either (a) purines labeled at the C-8 position and pyrimidines labeled at the C-5 and/or C-6 positions or (b) only C-6 labeled pyrimidines. There is no selective labeling of only certain purines or pyrimidines in this patent.
  • Stepwise oligonucleotide synthesis methods A variety of stepwise oligonucleotide synthesis methods employ repetition of steps including (a) blocking or protection of nucleotide sites in which no chemical moiety additions are desired; (b) exposure to the next desired nucleotide; and (c) removal of excess nucleotide and blocking agent.
  • Kaufman U.S. Patent No. 3,850,749 utilizes such a sequential synthesis with acyl nucleoside diphosphates added to an oligonucleotide.
  • Patents of Caruthers U.S. Patents No.
  • 4,415,732, 4,458,066 and 4,668,777 include use of trityl groups to block unreacted hydroxyl groups, formation of nucleoside phosphoramidite compounds that are activated by acidic compounds to permit reaction with a further nucleotide to yield a polynucleotide.
  • the patent of Koster provides for the preparation of oligonudeotides by (1) reaction of a nucleoside with a phosphine derivative; (2) reaction with a nucleoside bonded to a polymeric carrier; (3) oxidation of the carrier bound compound to form phosphotriester groups; (4) blocking of free primary 5'-hydroxy groups; (5) elimination of a protective group from the terminal 5'-hydroxy group of the developing oligomer; (6> subsequent repetition to form the desired oligonucleotide; and (7) elimination of the protective groups as appropriate.
  • Oligonucleotide synthesis of particular nucleotide sequences may be accomplished either manually or with DNA/RNA synthesizing machines.
  • Manual techniques generally employ solid phase techniques wherein solid supports, having a particular desired
  • SUBSTITUTESHEET 3'-end nucleotide attached are used to add nucleotides of the desired identity one by one.
  • certain similar protection and deprotection steps are used to ensure addition of the appropriate nucleotide to the oligonucleotide being synthesized.
  • Certain manual liquid phase DNA synthesis techniques are also used when large quantities of DNA are to be synthesized. See Li, 1987.
  • DNA synthesizing machines have made the preparation of oligonudeotides having known nucleotide percent compositions and sequences a rapid and reproducible technique.
  • the Applied. Biosystems Model 381A DNA Synthesizer uses as the chemistry of choice, the phosphoramidite method of oligonucleotide synthesis because of the inherently high coupling efficiencies and the stabilities of the starting materials.
  • the growing nucleotide chain is attached to a solid support derivatized with the nucleoside which is to be the 3'- hydroxyl end of the desired oligonucleotide product. The 5'- hydroxyl is blocked with a dimethoxytrityl group.
  • the support used for DNA synthesis is Controlled Pore Glass (CPG), a porous, non-swelling particle of 125-177 microns diameter and having 500 angstrom pores. Because of the attachment to the solid phase, excess reagents present in the liquid phase may be removed by filtration prior to addition of the next reagents without the need for purification steps between base additions.
  • CPG Controlled Pore Glass
  • the manufacturer's procedure of DNA synthesis on the Applied Biosystems machine involves the following steps:
  • SUBSTITUTESHEET end with the dimethoxytrityl group, with a weak acid, tetrazole, resulting in protonation of the amide-nitrogen of the phosphoramidite so that it is susceptible to nucleophilic attack; (3) Addition of the activated phosphoramidite derivative to the reaction vessel;
  • the manual and automatic oligonucleotide synthesis techniques that have been developed do not comprise methods of labeling particular single nucleotides within oligonudeotides. It is therefore an object of this invention to provide a means for synthesizing oligonudeotides that comprise a specific labeled nucleotide at a particular oligonucleotide site.
  • novel phosphoramidite compositions of this invention differ from compositions previously reported in that they comprise a tritium label at the l'-C and 2'-C position on the ribose or deoxyribose moiety.
  • the labeled oligonudeotides possess nucleotide sequences where the tritium-labeled nucleotide is positioned for study in biological applications.
  • nucleotide moieties that may be utilized in either tritium-labeled RNA or DNA sequences include: guanosine or deoxyguanosine, adenosine or deoxyadenosine, thymidine or deoxythymidine, cytidine or deoxycytidine, uridine or deoxyuridine.
  • the tritium-labeled positions in the above nucleotides may include replaceable hydrogen atoms of the structures shown in Figures 1 and 1A, where the letter "T" has been substituted on a ring system.
  • the tritium-labeled nucleotide was obtained from Amersham (Arlington Heights, IL) .
  • 1' ,2' [ 3 H]deox guanosine was used to synthesize oligonudeotides. It might also be possible to place a 3 H label on a purine or pyrimldine ring. Thus the C-8 hydrogen of purines could be replaced.
  • Figure 1 shows tritium label positions in representative nucleotides that may be used in the method of the invention.
  • Figure 1A shows tritium label positions in another nucleotide that may be used in the method of the invention.
  • Figure 2 is a diagram of the protected nucleotide used in oligonucleotide synthesis.
  • Figure 3 shows steps and products of Examples I-IV.
  • the present invention generally comprises radioactively- labeled phosphoramidites and polynucleotides and methods of synthesis and use thereof.
  • the synthesis steps to obtain the labeled phosphoramidites that are described in detail in Examples I-IV below are shown in Figure 3.
  • a preferred embodiment of this invention comprises tritium-labeled polydeoxyribonucleotides. Four such oligonudeotides were synthesized with 1' ,2*-[ 3 H]deoxyguanosine as illustrated in the following sequence: 5 ' -CC-G-T-G-G-G-ATATGGGCTG- 3 ' a b e d
  • A is a deoxyadenosine residue
  • C is a deoxycytidine residue
  • G is a deoxyguanosine residue
  • T is a deoxythymidine residue
  • a, b, c, and d are possible single sites of a tritium-labeled residue.
  • the tritium-labeled starting nucleotide, l',2'-[ 3 H] deoxyguanosine was protected by replacement of an active hydrogen atom of the pendant amino group on the purine base and hydrogen atoms of the hydroxyl groups on the deoxyribose nucleus as depicted in Figure 2 where ac is an acyl group such as isobutyryl; ak is an alkyl group such as trityl; and ph is a phosphoramidite group.
  • the method employed to obtain the compounds of the invention from the tritium-labeled nucleotide triphosphate comprises the following steps:
  • Trimethylsilyl groups are removed by the addition of concentrated ammonium hydroxide to yield the N- isobutyryl-[ 3 H]-nucleoside;
  • the nucleoside is tritylated (alkylation of a single hydroxyl group) at the 5 '-position of the deoxyribose methylol group with 4,4'-dimethoxytrityl hydrochloride (DMTr-Cl) in the presence of a 4- dimethylaminopyridine (DMAP) catalyst and triethylamine (TEA);
  • a manual or automatic DNA synthesizing technique for sequential addition of nucleotides is used to add the nucleotide moiety of the nucleoside phosphite to the oligonucleotide in the desired position or positions.
  • the actual synthesis of the desired polynucleotide sequence from selected nucleotides may be carried out by utilizing a DNA Synthesizer, such as the Applied Biosystems
  • nucleoside or deoxynucleoside residues for example, adenosine, cytidine, guanosine, uridine, thymidine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, and tritium-labeled nucleoside or deoxynucleoside (deoxyguanosine in the preferred embodiment of the invention) , are allowed to condense in a prescribed order.
  • the resultant oligonudeotides can be used, for example to determine the effects of chemical carcinogens or anti-cancer drugs on the primary DNA/RNA structure.
  • dG (280 mg) was first suspended in 7.5 ml of anhydrous pyridine and cooled to 4°C. Trimethylchlorosilane (520 ul) was added and the mixture stirred for 30 minutes and maintained at 4°C. Isobutyric anhydride (680 ul) was then added and the mixture stirred for 2 hours at room temperature. The reaction was cooled to 4°C and 2 ml of water added, followed 15 minutes later by addition of 1.8 ml of concentrated ammonium hydroxide. The sample was rotary evaporated to crystals and then a volume of 1-2 ml of water was added and the solution was extracted with 5 volumes of ether 2-3 times. The remaining aqueous phase was evaporated on a Savant Speed Vac (Savant Instruments, Inc., Farmingdale, NY) for 24 hours.
  • Savant Speed Vac Savant Instruments, Inc., Farmingdale, NY
  • the white powder (220 mg) was dissolved in methanol (5 ml) and loaded onto a liquid chromatographic column containing 30 cc of silica gel (Merck Keiselgel 60, 20-200 uM, EM Reagents, Darmstadt, Germany) and eluted with 16% methanol/methylene chloride (95% of the tritium counts came out in the first 70 ml).
  • the acylated product, 1 ' ,2' -[ 3 H]-N 2 -isobutyryl- deoxyguanosine (Product 1) was then rotary evaporated to dryness, transferred to a brown bottle, sealed with a rubber septum, and placed under vacuum for 48 hours to assure dryness.
  • the phosphoramidite was filtered on a glass sinter funnel to remove salts, dried on a rotary evaporator, dissolved in 3 ml ethyl acetate and precipitated by pipetting into 10 volumes of hexanes at-78°C.
  • Product 3 was redissolved in 2 ml acetonitrile, filtered through a 2 um filter into a brown vial and dried under vacuum for 24 hours.
  • nucleotides may be used in the procedure employed above to prepare acylated-alkoxylated-phosphitylated derivatives analogous to Product 3. Examples of other representative tritium-labeled starting nucleotides that may be used are shown in Figures 1 and 1A. EXAMPLE IV
  • Oligonucleotide synthesis and Isolation Product 3 was redissolved in anhydrous acetonitrile (3.5 ml) and placed on the Applied Biosystems 381A DNA Synthesizer.
  • Four separate oligonudeotides were synthesized with a l',2'[ 3 H]- deoxyguanosine (dG) located at only one of the deoxyguanosine sites as indicated with "a, b, c, or d" below:
  • This oligonucleotide sequence is a region of the hypoxanthine-guanine phosphoribosyl transferase gene where position "d” is a site frequently mutated by methylnitrosourea (MNU) as compared to positions "a", "b", or "c”_ Oligodeoxyribonucleotide synthesis, performed on the Applied Biosystems 381A DNA Synthesizer, had an overall coupling yield of 83% for the entire oligomer with a 90% coupling efficiency when the labeled deoxyguanosine phosphoramidite w as inserted.
  • the specific activities of the isolated four oligo ers were 10 uCi/mmol.
  • Preparation of a series of oligonudeotides that are identical except that a particular nucleotide (e.g., deoxyguanosine) is labeled at a different site in each oligonucleotide allows the study of the differential effects of various treatments (exposure to mutagens, various temperature and pH regimes , etc. ) on the same nucleotide located at different sites in the same oligonucleotide.
  • a particular nucleotide e.g., deoxyguanosine
  • the radioactively labeled polynucleotides of the invention comprise tritium-labeled polydeoxyribonucleotides.
  • the nucleotides of the invention are synthesized by the following steps: (l) acylation of a labeled nucleoside to form a first product; (2) tritylation of the first product to form a second product; (3) phosphitylation of the second product to form a labeled third phosphoramidite product; and (4) insertion of the third phosphoramidite product in an oligonucleotide at a predetermined site using a sequential oligonucleotide synthesis technique.
  • the invention provides a way to incorporate a radioactive label at a particular point in an oligonucleotide.
  • a radioactive label at a particular point in an oligonucleotide.
  • Such an oligonucleotide enables study of the ' effect of specific treatments, such as exposure to various chemicals, as well as enabling the specifically labeled reagent to be used to study cellular chemical phenomena.

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Abstract

On a synthétisé puis utilisé des phosphoramidites de nucléoside dans la préparation de nucléotides marqués au tritium sur des sites prédéterminés de polynucléotides ou d'oligonucléotides. On protège les nucléotides de purine et de pyrimidine marqués à des positions spécifiques au tritium sur les sites réactifs puis on les condense dans une séquence prédéterminée afin de produire des polynucléotides marqués au tritium. On peut utiliser les oligonucléotides marqués au tritium pour détermimer les effets de la modification de la structure d'ADN primaire sur la formation et la réparation des additifs d'ADN générés par des carcinogènes chimiques et des produits pharmaceutiques anticancéreux.
PCT/US1989/001535 1988-04-15 1989-04-12 Oligodesoxynucleotides marques au tritium specifiques a un site WO1989009780A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5316908A (en) * 1990-07-13 1994-05-31 Life Technologies, Inc. Size markers for electrophoretic analysis of DNA
US5486505A (en) * 1990-07-24 1996-01-23 Polygen Holding Corporation Polypeptide compounds having growth hormone releasing activity
US20190375774A1 (en) * 2016-11-23 2019-12-12 Wave Life Sciences Ltd. Compositions and methods for phosphoramidite and oligonucleotide synthesis

Citations (16)

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US3520872A (en) * 1967-03-24 1970-07-21 Upjohn Co Process for labelling purine and pyrimidine containing compounds
US3850749A (en) * 1971-06-16 1974-11-26 Yeda Res & Dev Preparation of oligoribonucleotides
US3852267A (en) * 1972-08-04 1974-12-03 Icn Pharmaceuticals Phosphoramidates of 3{40 ,5{40 -cyclic purine nucleotides
US4293652A (en) * 1979-05-25 1981-10-06 Cetus Corporation Method for synthesizing DNA sequentially
US4310662A (en) * 1979-12-26 1982-01-12 Genentech, Inc. Nucleosidic phosphorylating agent and methods
US4351901A (en) * 1980-03-24 1982-09-28 Cetus Corporation Method for single nucleotide alteration
US4415732A (en) * 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
US4458066A (en) * 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4652639A (en) * 1982-05-06 1987-03-24 Amgen Manufacture and expression of structural genes
US4668777A (en) * 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4672110A (en) * 1983-11-29 1987-06-09 Northwestern University Method of deprotecting polynucleotide trimethylethyl phosphotriesters
US4689405A (en) * 1983-01-20 1987-08-25 Gesellschaft Fur Biotechnologische Forschung Mbh (Gbf) Process for the simultaneous synthesis of several oligonucleotides on a solid phase
US4707352A (en) * 1984-01-30 1987-11-17 Enzo Biochem, Inc. Method of radioactively labeling diagnostic and therapeutic agents containing a chelating group
US4716106A (en) * 1984-03-01 1987-12-29 Amersham International Plc Detecting polynucleotide sequences
US4725677A (en) * 1983-08-18 1988-02-16 Biosyntech Gmbh Process for the preparation of oligonucleotides
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US3520872A (en) * 1967-03-24 1970-07-21 Upjohn Co Process for labelling purine and pyrimidine containing compounds
US3850749A (en) * 1971-06-16 1974-11-26 Yeda Res & Dev Preparation of oligoribonucleotides
US3852267A (en) * 1972-08-04 1974-12-03 Icn Pharmaceuticals Phosphoramidates of 3{40 ,5{40 -cyclic purine nucleotides
US4293652A (en) * 1979-05-25 1981-10-06 Cetus Corporation Method for synthesizing DNA sequentially
US4310662A (en) * 1979-12-26 1982-01-12 Genentech, Inc. Nucleosidic phosphorylating agent and methods
US4458066A (en) * 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4351901A (en) * 1980-03-24 1982-09-28 Cetus Corporation Method for single nucleotide alteration
US4668777A (en) * 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4415732A (en) * 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
US4652639A (en) * 1982-05-06 1987-03-24 Amgen Manufacture and expression of structural genes
US4689405A (en) * 1983-01-20 1987-08-25 Gesellschaft Fur Biotechnologische Forschung Mbh (Gbf) Process for the simultaneous synthesis of several oligonucleotides on a solid phase
US4725677A (en) * 1983-08-18 1988-02-16 Biosyntech Gmbh Process for the preparation of oligonucleotides
US4672110A (en) * 1983-11-29 1987-06-09 Northwestern University Method of deprotecting polynucleotide trimethylethyl phosphotriesters
US4707352A (en) * 1984-01-30 1987-11-17 Enzo Biochem, Inc. Method of radioactively labeling diagnostic and therapeutic agents containing a chelating group
US4792520A (en) * 1984-02-16 1988-12-20 University Of Cincinnati Methods and kits for identifying mutagenic agents and molecular mutations in DNA in mammalian cells
US4716106A (en) * 1984-03-01 1987-12-29 Amersham International Plc Detecting polynucleotide sequences

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Title
APPLIED BIOSYSTEMS MODEL 381A DNA SYNTHESIZER USERS MANUAL, Version 1.11, November 1985, Part No. A-900167-1, see pages 2-14 to 2-15. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 264, No. 2, issued January 1989 (Baltimore, MD) RICHARDSON et al.: Formation of O-methyldeoxyguanosine at specific sites in a synthetic oligonucleotide designed to resemble a known mutagenic hotspot, see pages 838-841. *
N.D. SINHA et al.: "Polymer support oligonucleotide synthesis XVIII: Use of B-cyanoethyl-N, N-dialkyl-/N morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product", IRL Press, published 1984, (Oxford, England), see pages 4554-4555. *
ROGER A. JONES: "Preparation of Protected Deoxyribonucleosides", in Oligonucleotides Synthesis. A Practical Approach. IRL Press, published 1984, (Washington, D.C.), see pages 23-27. *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5316908A (en) * 1990-07-13 1994-05-31 Life Technologies, Inc. Size markers for electrophoretic analysis of DNA
US5486505A (en) * 1990-07-24 1996-01-23 Polygen Holding Corporation Polypeptide compounds having growth hormone releasing activity
US20190375774A1 (en) * 2016-11-23 2019-12-12 Wave Life Sciences Ltd. Compositions and methods for phosphoramidite and oligonucleotide synthesis
US11873316B2 (en) * 2016-11-23 2024-01-16 Wave Life Sciences Ltd. Compositions and methods for phosphoramidite and oligonucleotide synthesis

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