WO1993019078A1 - Nucleotides modifies - Google Patents

Nucleotides modifies Download PDF

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Publication number
WO1993019078A1
WO1993019078A1 PCT/US1993/002390 US9302390W WO9319078A1 WO 1993019078 A1 WO1993019078 A1 WO 1993019078A1 US 9302390 W US9302390 W US 9302390W WO 9319078 A1 WO9319078 A1 WO 9319078A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
rhodamine
fluorescein
enzyme
jeffamine
Prior art date
Application number
PCT/US1993/002390
Other languages
English (en)
Inventor
Alberto Haces
Jeannette L. Flickinger
Jesse K. Mackey
Ayoub Rashtchian
Original Assignee
Life Technologies, 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 Life Technologies, Inc. filed Critical Life Technologies, Inc.
Priority to EP93907533A priority Critical patent/EP0638087A1/fr
Publication of WO1993019078A1 publication Critical patent/WO1993019078A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • the invention concerns novel compounds for nucleic acid labelling and methods for making nucleic acids incorporating the novel compounds.
  • Techniques for labelling nucleic acids with a reporter molecule generally fall into one of two categories: incorporating the label into the nucleic acid during synthesis, or post- synthetically modifying the nucleic acid.
  • the former is commonly accomplished by providing a labelled derivative of one of the precursor nucleotide triphosphates as a partial or complete substitute for the normal precursor during the enzyme-catalyzed synthesis of the nucleic acid.
  • the labelled nucleotide derivative must meet certain criteria. The derivative must continue to be recognized by the enzyme as a substrate, it must not interfere with or inhibit the enzyme and it must participate in the normal hydrogen-bonding interactions of base pairing, adenine with thy ine (or uracil) , guanine with cytosine.
  • Post-synthetic modification labelling is commonly accomplished by modifying the end group of a nucleic acid by means of a chemical reaction or by an enzyme such as terminal transferase.
  • reporter molecules have been incorporated into nucleic acids. Besides radioactive labels, which are usually incorporated during synthesis, fluorescent labels, chromatic labels, luminescent labels, ligands and haptens have been employed. Rhodamine and fluorescein have been used for fluorescent labelling. Nitroblue tetrazolium and BCIP (5-bromo- 4-chloro-3-indolylphosphate) (Gibco BRL) have been used as chromophores.
  • Firefly luciferin and PPD (4-methoxy-4-(3- phosphatenephenyl)spiro[l,2 dioxetane-3,2'-adamantane] have been used as luminescent labels.
  • Biotin has been used as a ligand to bind labelled streptavidin.
  • Dinitrophenol and digoxigenin have been used as hapten labels to bind antibody and take advantage of immunoassay methods.
  • the foregoing examples are illustrative only and not limiting.
  • Radioactive labels have the advantage of providing high sensitivity, however they have the disadvantages of being expensive, of having short shelf life in some cases, and of presenting safety and disposal problems. While non-radioactive labels lack the disadvantages of radioactivity, they pose other difficulties for the potential user. Many of the reporter compounds are large, bulky molecules relative to the nucleotides themselves, and their size can sterically interfere with incorporation. Low levels of incorporation cause loss of detection sensitivity by limiting the level of detectable signal per nucleic acid molecule.
  • the reaction conditions used for post-synthetic nucleic acid labelling can be incompatible with nucleic acid integrity and can involve specialized chemistry that requires equipment and reagents not normally found in the laboratories of many potential end users of the labelled nucleic acid.
  • linkers linear chains of, typically four to twelve atoms, usually a saturated or partially unsaturated aliphatic chain, occasionally containing an amide group.
  • the function of linkers has been considered to be to act as a spacer between the nucleotide base and the label. Any linkers capable of providing adequate spacing and flexibility have been considered functionally equivalent.
  • the use of linkers has also made possible a hybrid labelling technique whereby precursor nucleotides modified to possess a linker moiety are incorporated into the nucleic acid during synthesis, to yield linker-modified nucleic acid.
  • the linker • > ⁇ 5 groups suitable for such modification must have a reactive group at the free end of the linker chain.
  • the linker-modified nucleic acid is post-synthetically coupled with a reporter compound at the reactive ends of the incorporated linkers.
  • a reporter compound See, e.g., Jett et al., U.S. Application Serial Number 07/765,277).
  • the present invention is based on the discovery that Jeffamine (TM Texaco, Inc.)-linked nucleoside triphosphates can be substantially superior substrates for incorporating label into nucleic acids during enzyme-catalyzed synthesis. Use of a
  • novel Jeffamine-linked deoxy- or ribo- nucleotide derivatives are provided for any sort of nucleic acid label desired: fluorescent, chromatic, bio- or chemi- 25 luminescent, ligand or hapten.
  • Jeffamine-modified ribo- or deoxyribo-nucleotides are provided for synthesis of Jeffamine-modified nucleic acids. The latter can be post- synthetically modified by attachment of any desired label or combination of labels.
  • the invention further provides an improved method of
  • Jeffamines are polyglycol diamines having a general formula H 2 N-(CH 2 ) n -[0-(CH 2 ) n ] m -NH 2 , where n is 2 or more, preferably 2 to , and m is 1 to 10, preferably 1-5, most preferably 2 or 3. Where m is greater than l, there can be a different value of n for each [0-(CH 2 )]- group. However, typically and most conveniently, the value of n will be the same for each (CH 2 ) n group in the compound.
  • Useful Jeffamines of the invention are di-, tri- or tetra- and higher ethylene, n-propylene, or n- butylene glycol diamines. Although Jeffamines having branched CH 2 ⁇ n 9 rou P s are known, those preferred herein have linear (CH 2 ) n groups.
  • the structure of triethylene glycol diamine can be diagrammed as
  • One of the Jeffamine a ino groups is used to react with the base moiety of a nucleotide, the other amino group can reacted with a reporter moiety either before or after nucleic acid synthesis.
  • Reaction of a Jeffamine with adenine can be carried out at the 8 position or preferably, at the 6 position of adenine.
  • a Jeffamine derivative of guanine at the 8 position of guanine is the only suitable derivative known.
  • Cytosine can be reacted at the 4 position of cytosine.
  • Jeffamine derivatives of uracil and thymine are only feasible by indirect linkage. Such indirect linkage can be achieved, for example, by first forming allylamine derivatives of the pyrimidine ring at the 5 or 6 position, followed by subsequent reaction with a Jeffamine. Derivatives of cytosine can also be formed in like manner.
  • the labelled nucleotides of the invention therefore have three parts, the first being a deoxy- or ribo- nucleotide,
  • dN or rN abbreviated dN or rN (or d or rNTP if in the triphosphate form) which can be either d or r- adenosine (dA or rA) , d- or r- guanosine (dG or rG) or d- or r-cytosine (dC or rC) .
  • the second part is a Jeffamine-based linker, which will have a chain length of 7 or more atoms, depending on the Jeffamine used, and
  • n(J) where n is an integer of 7 or more.
  • the third part is the reporter, designated X.
  • X can be any molecule useful for labeling nucleic acid and having the ability to form stable compounds with a primary amine group.
  • X can be a fluorophore, for example, rhodamine or fluorescein.
  • X can be
  • a chromophore for example, Nitro blue tetrazolium, or BCIP.
  • X can be a luciferin or other luminescent reporter, such as PPD.
  • X can be a ligand, for example, biotin, having the property of binding another, readily detectable, molecule.
  • a ligand such as an enzyme cofactor can be detected by the activity
  • X can also be a hapten, for example dinitrophenol or digoxigenin, detectable by immunochemical means.
  • a labelled nucleotide of the invention is therefore 25 abbreviated herein as X-n(J)-(d or r)NTP.
  • modified nucleotide is defined herein as a nucleotide having a Jeffamine substituent but no reporter moiety (X is H) .
  • a modified nucleotide is useful for post-synthetic labelling of DNA in which the modified nucleotide has been
  • Such a modified nucleotide is abbreviated n(J)-(d or r)NTP, where n is 7 or more, and N is adenosine, guanosine or
  • Rho- 10(J)-dCTP was successfully added to DNA, using reaction conditions disclosed in U.S. Patent 4,878,979 for end-labelling DNA with Biotin-14-dATP.
  • Rhodamine-10(J)-dCTP Rhodamine-10(J)-dCTP and Fluorescein-10(J)-dCTP
  • the structures of Rhodamine-10(J)-dCTP and the corresponding Fluorescein-10(J)-dCTP are shown in Formula 1.
  • Formulas 2-5 show the structures of Fluorescein- and Rhodamine- 8-dATP, Fluorescein- and Rhodamine-4-dUTP, Rhodamine-8-dCTP, Fluorescein-(15)-dCTP, and Rhodamine-(15)-dCTP, respectively.
  • TEAB triethyl ammonium bicarbonate
  • the dye e.g., fluorescein or rhodamine
  • the reaction was monitored by thin layer chromatography (silica gel; butanol: acetone: acetic acid: 5% ammonium hydroxide: water/70:50:30:30:20) and/or by HPLC.
  • the crude mixtures were diluted in water (200 - 300 ml) , loaded on a 10 - 15 cm long, by 1 cm diameter column of mild anion exchange resins and eluted, sequentially, with 0.01, 0.2 and 0.5 M triethylammonium bicarbonate until the fraction containing the fluorescent dNTP was collected.
  • TLC, HPLC and capillary electrophoresis analysis was used to assess the purity and characteristic elution patterns of the desired product.
  • the compounds were characterized by their U.V. spectra as the overlapping spectra of the starting amino modified base and the dyes. Yields of fluorescent nucleotides were 50 - 60%.
  • the five fluorescent nucleotides were initially screened for enzymic incorporation into DNA using random primer extension with Klenow fragment of DNA polymerase I.
  • the five fluorescent nucleotides were fluorescein-8-dCTP, rhodamine-8 dATP, rhodamine- 8-dCTP, rhodamine-lO(J)-dCTP, and rhodamine-4-dUTP. All but rhodamine-lO(J) dCTP (which has two ether linkages) have alkylamine linkers.
  • Template DNA (100 ng) was denatured in a dilute buffer such as TE (10 M Tris-HCl, pH 7.5; 1 mM EDTA) by heating at 100°C for 5 min. Reaction components were added to final concentrations as follows: 50 mM Tris-HCl (pH 6.8), 5 mM MgCl 2 , 10 mM 2- mercaptoethanol, 400 ⁇ g/ml BSA, 300 ⁇ g/ml random octamers, 200 ⁇ M dCTP, dGTP, and dTTP,100 ⁇ M dCTP and 100 ⁇ M rhodamine-lO(J)-dCTP, 10 ⁇ Ci ⁇ -[ 32 P]- dATP (3000Ci/mmol) and 40 units Klenow fragment in a final volume of 50 ⁇ l.
  • TE Tris-HCl
  • pH 7.5 pH 7.5
  • EDTA 10 M Tris-HCl
  • 2- mercaptoethanol 400
  • Rhodamine-8-dCTP 0.1 13 Rhodamine-10(J)-dCTP 42.6 w 5623
  • Template DNA 500 ng was denatured in a dilute buffer such as TE (10 mM Tris-HCl, pH 7.5; 1 mM EDTA) by heating at 100°C for 10 min. Reaction components were added to final concentrations as follows: 50 mM Tris-HCl (pH 6.8), 5 mM MgCl 2 , 10 mM 2- mercaptoethanol, 400 ⁇ g/ml BSA, 300 ⁇ m/ml random octamers, 100 ⁇ M each of dATP, dGTP, dTTP, and dCTP, 10 ⁇ Ci ⁇ -[ 32 P]-dATP (3000 Ci/mmol) , and 40 units Klenow fragment in a final volume of 50 ⁇ l.
  • TE Tris-HCl
  • pH 7.5 pH 7.5
  • EDTA mM 2- mercaptoethanol
  • 400 ⁇ g/ml BSA 300 ⁇ m/ml random octamers
  • the corresponding unmodified dNTP was replaced with 100 ⁇ M of the modified nucleoside triphosphates.
  • different mixtures of modified and unmodified nucleotides were used.
  • the percentage of modified nucleotides to the total modified plus corresponding unmodified nucleotide was varied from 25% to 100%. IN all cases, the total concentration of each nucleotide was kept at 100 ⁇ M which resulted in a total nucleotide concentration of 400 ⁇ M. After incubation at 37°C for 2 hours, 5 ⁇ l 0.2 M EDTA (pH 7.5) was added to terminate the reaction.
  • Incorporation was determined by trichloroacetic acid (TCA) precipitation. Diluted aliquots from the reaction were spotted on glass fiber filters in duplicate and dried. One of the duplicate filters was washed four times in cold 5% TCA, 20 mM sodium pyrophosphate, then rinsed in 70% ethanol and dried and counted in a liquid scintillation counter (incorporated counts) . The second filter was counted directly in the scintillation counter (total counts) . Incorporation of radioactive label was used to determine synthesis of fluorescent DNA probe. When the modified nucleotide was a dATP derivative, ⁇ -[ 32 P]-dCTP was used as a trace label.
  • TCA trichloroacetic acid
  • Table 3 is a list of additional modified nucleotides that have been screened for use in enzymatic incorporation into DNA using random primer extension with Klenow fragment of DNA polymerase.
  • Rhodamine-10(J)-dCTP Fluorescein-10(J)-dCTP Fluorescein-(15)-dCTP Fluorescein-4-dUTP Rhoda ine-(12)-dUTP

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne de nouveaux composés de nucléotides modifiés représentés par la formule: X-n(J)-(d ou r)NTP. Dans cette formule, N représente adénosine, guanosine ou cytidine; X représente H, un fluorophore, un chromophore, un composé luminescent, un ligand ou un haptène; n représente un entier supérieur ou égal à 7; et J représente un substituant de Jeffamine (TM Texaco, Inc.). Ces composés constituent des substrats sensiblement supérieurs pour incorporer une étiquette dans des acides nucléiques pendant la synthèse par catalyse enzymatique. L'utilisation d'un dérivé de nucléotide étiqueté lié par la Jeffamine en tant que substituant partiel pour un substrat non étiqueté permet par conséquent d'obtenir une meilleure incorporation et donc une meilleure densité d'étiquette par unité de longueur d'acide nucléique que celle obtenue avec la même étiquette fixée au moyen d'un agent de liaison de la technique antérieure. Des ribonucléotides ou des désoxyribonucléotides modifiés par la Jeffamine sont également utilisés pour synthétiser des acides nucléiques modifiés par la Jeffamine. Cette invention concerne également un procédé amélioré de synthèse d'acides nucléiques étiquetés. Ce procédé assure une meilleure fréquence et une plus grande efficacité de l'incorporation d'étiquette et nécessite donc des quantités plus faibles de dérivé de nucléotide dans le mélange de réaction.
PCT/US1993/002390 1992-03-17 1993-03-17 Nucleotides modifies WO1993019078A1 (fr)

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EP93907533A EP0638087A1 (fr) 1992-03-17 1993-03-17 Nucleotides modifies

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US85268892A 1992-03-17 1992-03-17
US07/852,688 1992-03-17

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1152010A2 (fr) * 2000-05-05 2001-11-07 Wallac Oy Oligonucléotide agent de marquage et leur utilisation
US6811979B2 (en) * 2000-10-11 2004-11-02 Applera Corporation Fluorescent nucleobase conjugates having anionic linkers
FR2893617A1 (fr) * 2005-11-24 2007-05-25 Inst Nat Sante Rech Med Analogues photoactivables du nadh, du nadph, du nad+ ou du nadp+

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4828979A (en) * 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
US4914210A (en) * 1987-10-02 1990-04-03 Cetus Corporation Oligonucleotide functionalizing reagents
US4962029A (en) * 1987-10-02 1990-10-09 Cetus Corporation Covalent oligonucleotide-horseradish peroxidase conjugate

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0407816A3 (en) * 1989-07-14 1993-01-27 Abbott Laboratories Base modified nucleosides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4828979A (en) * 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
US4914210A (en) * 1987-10-02 1990-04-03 Cetus Corporation Oligonucleotide functionalizing reagents
US4962029A (en) * 1987-10-02 1990-10-09 Cetus Corporation Covalent oligonucleotide-horseradish peroxidase conjugate

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Electrophoresis, Volume 13, issued September 1992, W. ANSORGE et al., "High Throughput Automated DNA Sequencing Facility with Fluorescent Labels at the European Molecular Biology Laboratory", pages 616-619, see especially page 617. *
Journal of Cellular Biochemistry, Supplement 16B, issued February 1992, M.L. HAMMOND et al., "Enzymatic Synthesis and Exonucleolytic Degradation of Fluorescent DNA Containing Rhodamine and Fluorescein Nucleotides", page 46, Abstract F325, see entire Abstract. *
Methods in Molecular and Cellular Biology, Volume 3, issued February 1992, H. VOSS et al., "New Procedure for Automated DNA Sequencing with Multiple Internal Labeling by Fluorescent dUTP", pages 30-34, see especially page 33. *
Proceedings National Academy of Sciences, USA, Volume 78, No. 11, issued November 1981, P.R. LANGER et al., "Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes", pages 6633-6637, see entire document. *
See also references of EP0638087A4 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1152010A2 (fr) * 2000-05-05 2001-11-07 Wallac Oy Oligonucléotide agent de marquage et leur utilisation
EP1152010A3 (fr) * 2000-05-05 2002-01-02 Wallac Oy Oligonucléotide agent de marquage et leur utilisation
US6949639B1 (en) 2000-05-05 2005-09-27 Wallac Oy Oligonucleotide labeling reactants and their use
US6811979B2 (en) * 2000-10-11 2004-11-02 Applera Corporation Fluorescent nucleobase conjugates having anionic linkers
US7429651B2 (en) 2000-10-11 2008-09-30 Applera Corporation Fluorescent nucleobase conjugates having anionic linkers
US7687236B2 (en) 2000-10-11 2010-03-30 Applied Biosystems, Llc Fluorescent nucleobase conjugates having anionic linkers
FR2893617A1 (fr) * 2005-11-24 2007-05-25 Inst Nat Sante Rech Med Analogues photoactivables du nadh, du nadph, du nad+ ou du nadp+

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Publication number Publication date
EP0638087A4 (fr) 1994-12-27
CA2117583A1 (fr) 1993-09-30
EP0638087A1 (fr) 1995-02-15

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