Saccharide specific binding system labeled nucleotides

• US 5,260,433 A
• Filed: 08/13/1990
• Issued: 11/09/1993
• Est. Priority Date: 06/23/1982
• Status: Expired due to Term

• Current Assignee: Enzo Diagnostics, Inc.
• Original Assignee: Enzo Diagnostics, Inc.
• Inventors: Stavrianopoulos, Jannis G., Rabbani, Elazar, Kirtikar, Dollie, Kline, Stanley, Engelhardt, Dean
• Primary Examiner(s): Rollins, John W.
• Application Number:
  US07/567,039
• Time in Patent Office: 1,184 Days
• Field of Search: 435/6, 536/27
• US Class Current: 536/23.1
• 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

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 selected from the group consisting of (i) a ribonucleotide having the formula, ##STR1## wherein PM is a phosphate moiety, SM is a sugar moiety, BASE is a pyrimidine, purine or 7-deazapurine moiety, and Sig is a saccharide moiety. PM is attached at the 2'"'"',3'"'"' or 5'"'"' position of SM, BASE is attached to the 1'"'"' position of SM from the N¹ position when BASE is a pyrimidine or covalently attached to SM; and (ii) a nucleotide having the formula, ##STR2## wherein PM, SM, BASE and Sig are as defined above but PM is attached to the 3'"'"' or the 5'"'"' position of SM when said nucleotide is a deoxyribonucleotide and at the 2'"'"', 3'"'"' or 5'"'"' position when said 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, and wherein Sig is covalently attached to PM.

Claims

1. A nucleotide selected from the group consisting of:

(i) a ribonucleotide having the formula, ##STR21## wherein PM is a phosphate moiety,

SM is a sugar moiety,

BASE is a pyrimidine, purine or 7-deazapurine moiety, and

Sig is a detectable saccharide moiety,

PM is attached at the 2'"'"', 3'"'"' or 5'"'"' position of SM, 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 7-deazapurine; and

Sig is covalently attached to SM; and

(ii) a nucleotide having the formula;

##STR22## wherein PM is a phosphate moiety,

SM is a sugar moiety,

BASE is a pyrimidine, purine or 7-deazapurine moiety,

Sig is a detectable saccharide moiety,

PM is attached at the 3'"'"' or the 5'"'"' position of SM when said nucleotide is a deoxyribonucleotide and at the 2'"'"', 3'"'"' or 5'"'"' position when said 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, and wherein Sig is covalently attached to PM.

2. The nucleotide of claim 1 wherein the nucleotide has the formula ##STR23##

3. A polynucleotide comprising at least one nucleotide in accordance with claim 2.

4. A polynucleotide comprising at least one nucleotide in accordance with claim 2.

5. The nucleotide of claim 1 wherein the nucleotide has the formula ##STR24##

6. The nucleotide of claim 1 wherein the saccharide moiety is a monosaccharide.

7. The nucleotide according to claim 1 wherein Sig is attached to the nucleotide directly or through a linkage group which linkage group does not interfere substantially with the characteristic ability of Sig to form a detectable signal and which Sig is detectable when an oligo- or polynucleotide containing the nucleotide is incorporated into a double-stranded ribonucleic or deoxyribonucleic acid duplex.

8. The nucleotide according to claim 1 wherein Sig is covalently attached to PM via the chemical linkage ##STR25##

9. A nucleotide in accordance with claim 1 wherein Sig is a monosaccharide, a polysaccharide or an oligosaccharide.

10. A nucleotide in accordance with claim 1 wherein Sig is a sugar selected from the group consisting of triose, tetrose, pentose, hexose, heptose and octose.

11. A nucleotide in accordance with claim 1 wherein Sig includes a glycosidic linkage moiety.

12. A nucleotide in accordance with claim 1 wherein Sig is a hexose moiety.

13. A nucleotide in accordance with claim 1 wherein Sig is a sugar residue and wherein said sugar residue is attached to a saccharide binding protein.

14. A nucleotide in accordance with claim 13 wherein said protein is a lectin.

15. A nucleotide in accordance with claim 13 wherein said protein is a lectin.

16. A nucleotide in accordance with claim 15 wherein said lectin is Concanavalin A.

17. A nucleotide in accordance with claim 14 wherein said lectin is a plant lectin selected from the group consisting of Concanavalin A, LCH lentil lectin, PSA pea lectin and BFA Vicia Faba lectin.

18. A nucleotide in accordance with claim 1 wherein Sig comprises an α

-mannosyl residue and wherein said α

-mannosyl residue is bound to Concanavalin A.

19. A nucleotide in accordance with claim 1 wherein Sig comprises an N-acetylglucosamine residue and wherein the N-acetylglucosamine residue is bound to wheat germ agglutinin.

20. A polynucleotide comprising at least one nucleotide in accordance with claim 1.

21. A nucleotide in accordance with claim 1 wherein Sig comprises a component selected from the group consisting of a saccharide component, a protein component capable of binding to said saccharide component and a glycosylated enzyme component capable of binding to said protein component.

22. A nucleotide in accordance with claim 21 wherein said protein component is a lectin.

23. A nucleotide in accordance with claim 21 wherein said enzyme component is an enzyme selected from the group consisting of alkaline phosphatase, acid phosphatase and horseradish peroxidase.

24. A nucleotide in accordance with claim 1 wherein said saccharide component is a monosaccharide, oligosaccharide or polysaccharide.