Labeling reagents and labeled targets, target labeling processes and other processes for using same in nucleic acid determinations and analyses

• US 7,166,478 B2
• Filed: 03/12/2002
• Issued: 01/23/2007
• Est. Priority Date: 03/12/2002
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Rabbani, Elazar
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/096,075
• Publication Number:
  US 20030225247A1
• Time in Patent Office: 1,778 Days
• Field of Search: 435/6, 536/23.1, 536/25.3, 536/26.6, 436/544
• US Class Current: 436/544
• 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
• CPC Class Codes: C09B 47/00 Porphines; Azaporphines non-dyeing compounds C07D487/22
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

This invention provides for labeling reagents, labeled targets and processes for preparing labeling reagents. The labeling reagents can take the form of cyanine dyes, xanthene dyes, porphyrin dyes, coumarin dyes or composite dyes. These labeling reagents are useful for labeling probes or targets, including nucleic acids and proteins. These reagents can be usefully applied to protein and nucleic acid probe based assays. They are also applicable to real-time detection processes.

Claims

1. A labeling reagent for labeling a target, said labeling reagent comprising a marker moiety M and a reactive group R

M-R

wherein said marker moiety M and said reactive group R are covalently linked together by a linker arm comprising a backbone which comprises one or more carbon atoms, wherein said backbone further comprises one or more peptide bonds, said M comprising at least one moiety that comprises a ligand, a dye, or both a ligand and a dye;

and wherein said target is labeled by forming a carbon-carbon bond between a carbon atom in said target and a carbon atom in said labeling reagent by means of said reactive group R.

2. The labeling reagent of claim 1, wherein said target comprises a protein, a peptide, a nucleic acid, a nucleotide or a nucleotide analog, a receptor, a natural or synthetic drug, a synthetic oligomer, a synthetic polymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, an antigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- or polysaccharide.

3. The labeling reagent of claim 2, wherein said nucleic acid or nucleotide or nucleotide analog is modified.

4. The labeling reagent of claim 2, wherein said ligand comprises biotin, iminobiotin, digoxygenin or fluorescein.

5. The labeling reagent of claim 1, wherein said dye comprises a fluorescent dye.

6. The labeling reagent of claim 5, wherein said fluorescent dye comprises a xanthene dye, a cyanine dye, a coumarin dye, a porphyrin dye or a composite dye.

7. The labeling reagent of claim 6, wherein said xanthene dye comprises fluorescein, a substituted fluorescein, rhodamine, a substituted rhodamine, rhodol, or a substituted rhodol.

8. The labeling reagent of claim 1, wherein said linker arm is flexible, semi-rigid or rigid.

9. The labeling reagent of claim 1, wherein said linker arm has a backbone comprising at least one non-carbon atom.

10. The labeling reagent of claim 9, wherein said non-carbon atom comprises sulfur, oxygen or nitrogen.

11. The labeling reagent of claim 1, wherein said reactive group R comprises an alkene group, an alkyne group, a halogenated compound or a metallo-organic compound.

12. The labeling reagent of claim 11, wherein said metallo-organic compound comprises mercury, zinc, copper or platinum.

13. The labeling reagent of claim 11, wherein said metallo-organic compound comprises an aromatic group, an aromatic heterocyclic group, an alkene group or an alkyne group.

14. A process for labeling a target, said process comprising the steps of:

(a) providing;

(i) said target; and

(ii) a labeling reagent for labeling a target, said labeling reagent comprising a marker moiety M and a reactive group R

M-R

wherein said marker moiety M and said reactive group R are covalently linked together by a linker arm comprising a backbone which comprises one or more carbon atoms comprising one or more peptide bonds, said M comprising at least one moiety that comprises a ligand, a dye, or both a ligand and a dye; and

said reactive group R forming a carbon-carbon linkage with said target under appropriate conditions; and

(b) reacting said target (i) and said labeling reagent (ii) under conditions such that a carbon-carbon linkage forms between said target (i) and said labeling reagent (ii), thereby labeling said target (i) with said marker moiety M.

15. The process of claim 14, wherein in said providing step, the target comprises a protein, a peptide, a nucleic acid, a nucleotide or a nucleotide analog, a receptor, a natural or synthetic drug, a synthetic oligomer, a synthetic polymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, an antigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- or polysaccharide.

16. The process of claim 14, wherein in said providing step, the ligand comprises biotin, iminobiotin, digoxygenin or fluorescein, and the dye comprises a fluorescent dye.

17. The process of claim 16, wherein said fluorescent dye comprises a xanthene dye, a cyanine dye, a coumarin dye, a porphyrin dye or a composite dye.

18. The process of claim 14, wherein said providing or reacting step, the reactive group R comprises an alkene group, an alkyne group, a halogenated compound or a metallo-organic compound.

19. The process of claim 14, wherein in said providing step, the target has been modified.

20. The process of claim 19, wherein said modified target comprises an alkene group, an alkyne group, a halogenated compound or a metallo-organic compound.