Efficient activated cyanine dyes

• US 6,110,630 A
• Filed: 06/18/1998
• Issued: 08/29/2000
• Est. Priority Date: 06/18/1998
• Status: Expired due to Term

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Inventors: Hanna, Naeem B., Farooqui, Firdous, Reddy, M. Parameswara, Michael, Maged A.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/100,150
• Time in Patent Office: 803 Days
• Field of Search: 435/6, 430/581, 430/93, 436/519, 536/25.32, 548/427
• US Class Current: 430/93
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups

Abstract

Activating groups based on N-hydroxynaphthalimide, are disclosed herein. The activating groups can mediate the coupling of labeling moieties, such as biotin or cyanine dyes, to a variety of components, including chain terminators, nucleoside triphosphates, and oligonucleotides, which are used in nucleotide sequencing. From these activating groups, activated esters of the labeling moieties can be prepared. The activated esters react with a component, for example a derivatized nucleotide chain terminator, to give a labeled component. In additions, methods of the present invention provide for labeling a nucleoside triphosphate in organic media. The activating groups and methods of the present invention allow the activation and coupling reactions to occur at a much higher yield, compared with the prior art.

Claims

1. An activated dye having the formula:

##STR7## wherein n is 1, 2, or 3, X is S, O, N, CH₂, or C(CH₃)₂, R1 is N-oxynaphthalimide-Rx, R2 is alkyl, alkylsulfonate, alkylcarboxylate, activated alkylsulfonate, or activated alkylcarboxylate having an alkyl chain about one to ten carbon atoms long,

whereby Rx is selected from the group consisting of hydrogen, sulfonyl, and alkyl.

2. A method of preparing an activated dye, the method comprising the steps of:

dissolving a cyanine dye having at least one carboxyl group in a solvent; and

combining N-hydroxynaphthalimide-Rx, wherein Rx is selected from the group consisting of hydrogen, sulfonyl, and alkyl, and an activating agent, with the dissolved cyanine dye to form an activated dye.

3. The method of claim 2, wherein the cyanine dye is selected from the group consisting of Cy5, benzo-Cy5, dibenzo-Cy5, Cy7, benzo-Cy7, and dibenzo-Cy7.

4. The method of claim 2, wherein the solvent comprises an organic solvent.

5. The method of claim 2, wherein the solvent comprises dimethyformamide.

6. The method of claim 2, wherein the amount of N-hydroxynaphthalimide combined with the cyanine dye is about two or more molar equivalents.

7. The method of claim 6, wherein the amount of N-hydroxynaphthalimide-Rx ranges from about 1.5 to about 5 molar equivalents.

8. The method of claim 2, further comprising the additional step of precipitating the activated dye from the mixture.

9. The method of claim 8, wherein the activated dye is precipitated by adding ethyl acetate.

33. The method of claim 2, wherein the activating agent is selected from the group consisting of carbonyl diimidazole and dicyclohexylcarbodiimide.

10. A method of labeling an oligonucleotide, the method comprising the steps of:

modifying an oligonucleotide to contain a nucleophile thereby forming a modified oligonucleotide;

combining the modified oligonucleotide with an activated dye having at least one N-hydroxynaphthalimide-Rx group, wherein Rx is selected from the group consisting of hydrogen, sulfonyl, and alkyl.

11. The method of claim 10, wherein the nucleophile is an amino or thiol group.

12. The method of claim 10 wherein the modified oligonucleotide is an amino-oligonucleotide.

13. The method of claim 10 wherein the terminus of the oligonucleotide is modified.

14. The method of claim 10, wherein the activated dye is selected from the group consisting of:

##STR8## wherein n is 1, 2, or 3, X is S, O, N, CH₂, or C(CH₃)₂, R1 is N-oxynaphthalimide-Rx, R2 is alkyl, alkylsulfonate, alkylcarboxylate, activated alkylsulfonate, or activated alkylcarboxylate having an alkyl chain about one to ten carbon atoms long,

whereby Rx is selected from the group consisting of hydrogen, sulfonyl, and alkyl.

15. The method of claim 10, wherein the activated dye is combined in molar excess relative to the modified nucleotide.

16. A method of labeling a component comprising,

combining the component with an activated moiety having at least one N-hydroxynapthalimide-Rx group, wherein Rx is selected from the group consisting of hydrogen, sulfonyl and alkyl.

17. The method of claim 16, wherein the component is selected from the group consisting of antibodies, proteins, peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, antigens, haptens, lectins, avidin, streptavidin, toxins, carbohydrates, oligosaccharides, polysaccharides, and other materials including nucleic acids, DNA, derivatized nucleic acids, derivatized deoxynucleic acids, DNA fragments, RNA fragments, derivatized DNA fragments, derivatized RNA fragments, drugs, toxins, blood cells, microbial materials, particles, plastic or glass surfaces, polymer membranes, nucleotides, deoxynucleotides, and dideoxynucleotides.

18. The method of claim 16, wherein the activated moiety is selected from the group consisting of biotin, hapten, cyanine dyes, merocyanine dyes, styryl dyes, fluorophores, fluorescein, rhodamine, Texas red, chemiluminescent molecules, acridinium esters, dioxitane derivatives, electroluminescent labels, ruthenium based molecules, hapten molecules, digoxigenin, chromophore molecules and coumarins.

19. A method of labeling a nucleoside triphosphate comprising, combining a nucleoside triphosphate having a nucleophilic group with an activated moiety having at least one N-hydroxynapthalimide-Rx group, wherein Rx is selected from the group consisting of hydrogen, sulfonyl and alkyl.

20. The method of claim 19, wherein the nucleophilic group is an amino group.

21. The method of claim 19, wherein the activated moiety is selected from the group consisting of biotin, hapten, cyanine dyes, merocyanine dyes, styryl dyes, fluorophores, fluorescein, rhodamine, Texas red, chemiluminescent molecules, acridinium esters, dioxitane derivatives, electroluminescent labels, ruthenium based molecules, hapten molecules, digoxigenin, chromophore molecules and coumarins.

22. A method of labeling a nucleoside triphosphate, the method comprising the steps of:

converting the nucleoside triphosphate to its trialkyl ammonium salt to form a nucleoside triphosphate trialkyl ammonium salt; and

combining the nucleoside triphosphate trialkyl ammonium salt with an activated moiety having an activated ester group.

23. The method of claim 22, wherein the trialkyl ammonium salt is a tributyl ammonium salt.

24. The method of claim 22, wherein the activated ester group is selected from the group consisting of N-hydroxysuccinimide, paranitrophenol, N-hydroxyphthalimide, and N-hydroxynaphthalimide-Rx activated esters, wherein Rx is selected from the group consisting of hydrogen, sulfonyl and alkyl.

25. The method of claim 22, wherein the activated moiety is selected from the group consisting of biotin, hapten, cyanine dyes, merocyanine dyes, styryl dyes, fluorophores, fluorescein, rhodamine, Texas red, chemiluminescent molecules, acridinium esters, dioxitane derivatives, electroluminescent labels, ruthenium based molecules, hapten molecules, digoxigenin, chromophore molecules and coumarins.

26. The method of claim 22, wherein the method is performed in an organic solvent.

27. A method of preparing a dye labeled dideoxynucleotide triphosphate terminator, the method comprising step of:

combining in a reaction mixture, a tributyl ammonium salt of a triphosphate nucleotide terminator; and

an activated dye having at least one N-hydroxynapthalimide-Rx group.

28. A method of labeling an oligonucleotide, the method comprising the steps of:

converting the oligonucleotide to its trialkyl ammonium salt to form an oligonucleotide trialkyl ammonium salt; and

combining the oligonucleotide trialkyl ammonium salt with an activated moiety having at least one activated ester group.

29. The method of claim 28, wherein the activated moiety is selected from the group consisting of biotin, hapten, cyanine dyes, merocyanine dyes, styryl dyes, fluorophores, fluorescein, rhodamine, Texas red, chemiluminescent molecules, acridinium esters, dioxitane derivatives, electroluminescent labels, ruthenium based molecules, hapten molecules, digoxigenin, chromophore molecules and coumarins.

30. The method of claim 28, wherein the method is performed in an organic solvent.

31. The method of claim 28, wherein the trialkyl ammonium salt is a tributyl ammonium salt.

32. The method of claim 28, wherein the activated ester group is selected from the group consisting of N-hydroxysuccinimide, paranitrophenol, N-hydroxyphthalimide, and N-hydroxynaphthalimide-Rx activated esters, wherein Rx is selected from the group consisting of hydrogen, sulfonyl and alkyl.