Base moiety-labeled detectable nucleatide

• US 5,241,060 A
• Filed: 06/04/1990
• Issued: 08/31/1993
• Est. Priority Date: 06/23/1982
• Status: Expired due to Term

• Current Assignee: Enzo Diagnostics, Inc.
• Original Assignee: Enzo Diagnostics, Inc.
• Inventors: Kirtikar, Dollie, Engelhardt, Dean, Stavrianopoulos, Jannis G., Rabbani, Elazar, Kline, Stanley
• Primary Examiner(s): Rollins, John W.
• Application Number:
  US07/532,704
• Time in Patent Office: 1,184 Days
• Field of Search: 536/27
• US Class Current: 536/25.32
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07K 17/06 attached to the carrier via a bridging agent
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q

Abstract

The present invention provides nucleotides and polynucleotides which are chemically modified or labeled so as to be capable of ready detection when attached to and/or incorporated in nucleic acid material. More particularly, this invention provides a nucleotide having the formula

PM-SM-BASE-Sig

wherein PM is a phosphate moiety, SM is a sugar moiety and BASE is a pyrimidine, purine or 7-deazapurine moiety. PM is attached at the 3'"'"' or the 5'"'"' position of SM when the nucleotide is a deoxyribonucleotide and at the 2'"'"', 3'"'"' or 5'"'"' position when the nucleotide is a ribonucleotide. BASE is attached to the 1'"'"' position of SM from the N¹ position when BASE is a pyrimidine or the N⁹ position when BASE is a purine or a 7-deazapurine. Sig is a detectable moiety that is covalently attached to BASE at a position other than the C⁵ position when BASE is a pyrimidine, at a position other than the C⁸ position when BASE is a purine and at a position other than the C⁷ position when BASE is a 7-deazapurine.

Claims

1. A nucleotide having the formula PM-SM-BASE-Sig wherein PM is a phosphate moiety, SM is a sugar moiety, BASE is a pyrimidine, purine or 7-deazapurine moiety, PM being attached at the 3'"'"' or the 5'"'"' position of SM when the nucleotide is a deoxyribonucleotide and at the 2'"'"', 3'"'"' or 5'"'"' position when the nucleotide is a ribonucleotide, BASE being attached to the 1'"'"' position of SM from the N¹ position when BASE is a pyrimidine or the N⁹ position when BASE is a purine or a 7-deazapurine, and Sig is covalently attached to BASE at a position other than the C⁵ position when BASE is a pyrimidine, at a position other than the C⁸ position when BASE is a purine and at a position other than the C⁷ position when BASE is a 7-deazapurine and wherein Sig represents a detectable moiety.

2. An oligo- or polydeoxyribonucleotide comprising at least one nucleotide in accordance with claim 1.

3. An oligo- or polyribonucleotide comprising at least one nucleotide in accordance with claim 1.

4. A nucleotide in accordance with claim 1 wherein Sig is a moiety containing at least 3 carbon atoms.

5. The nucleotide of claim 1 wherein Sig is selected from the group consisting of mono-, oligo- and polysaccharides.

6. The nucleotide of claim 5 wherein Sig is selected from the group consisting of triose, tetrose, pentose, hexose, heptose and octose.

7. The nucleotide of claim 1 wherein Sig includes a glycosidic linkage moiety.

8. The nucleotide of claim 1 wherein Sig is a sugar residue and such sugar residue is complexed with a binding protein for such sugar residue.

9. The nucleotide of claim 8 wherein such binding protein is a lectin.

10. The nucleotide of claim 9 wherein such lectin is Concanavalin A.

11. The nucleotide of claim 10 wherein said Concanavalin A is conjugated to ferritin.

12. The nucleotide of claim 9 wherein such lectin is conjugated to ferritin.

13. The nucleotide of claim 1 wherein Sig comprises a component selected from the group consisting of biotin, iminobiotin, an electron dense component, a magnetic component, an enzyme, a hormone component, a radioactive component, a metal-containing component, a fluorescent component, a chemiluminescent component an antigen, a hapten and an antibody component.

14. The nucleotide of claim 13 wherein such electron dense component is ferritin.

15. The nucleotide of claim 13 wherein Sig comprises a radioactive isotope.

16. The nucleotide of claim 15 wherein such radioactive isotope is radioactive cobalt.

17. The nucleotide of claim 13 wherein Sig comprises an enzyme.

18. The nucleotide of claim 17 wherein such enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, B-galactosidase, ribonuclease, glucose oxidase and peroxidase.

19. The nucleotide of claim 13 wherein Sig comprises a fluorescent component.

20. The nucleotide of claim 19 wherein such fluorescent component is selected from the group consisting of fluorescein, rhodamine and dansyl.

21. The nucleotide of claim 13 wherein Sig comprises a magnetic component.

22. The nucleotide of claim 21 wherein such magnetic component comprises a magnetic oxide.

23. The nucleotide of claim 22 wherein such magnetic oxide is ferric oxide.

24. The nucleotide of claim 13 wherein Sig includes a hapten component capable of complexing with an antibody specific thereto.

25. The nucleotide of claim 1 wherein Sig includes a catalytic metal-containing component.

26. An oligo- or polynucleotide comprising at least one nucleotide of claim 1 and wherein the oligo- or polynucleotide is terminally ligated or attached to a polypeptide.

27. A composition comprising an oligo-or polynucleotide including at least one nucleotide of claim 1, a polypeptide capable of forming a complex with Sig and a moiety which can be detected when such complex is formed.

28. The composition of claim 27 wherein such polypeptide comprises a polylysine.

29. The composition of claim 27 wherein such polypeptide is selected from the group consisting of at least one of avidin, streptavidin and anti-Sig immunoglobulin.

30. The composition of claim 27 wherein Sig is a ligand and such polypeptide is an antibody thereto.

31. The composition of claim 27 wherein said detectable moiety is selected from the group consisting of biotin, iminobiotin, an electron dense component, a magnetic component, an enzyme, a hormone component, a radioactive component, a metal-containing component, a fluorescent component, a chemiluminescent component an antigen, a hapten and an antibody component.