Nucleic acid binding compounds containing pyrazolo[3,4-D]pyrimidine analogues of purin-2,6-diamine and their uses

• US 8,057,997 B2
• Filed: 04/30/2007
• Issued: 11/15/2011
• Est. Priority Date: 08/03/2000
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Seidel, Christoph, Heindl, Dieter, Von Der Eltz, Herbert, Becher, Georg, Seela, Frank, Bergmann, Frank
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/742,416
• Publication Number:
  US 20110021365A1
• Time in Patent Office: 1,660 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 435/6
• CPC Class Codes: C07H 19/16 Purine radicals
• 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: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is in the field of nucleic acid binding compounds comprising 7-substituted 7-deaza-8aza-2,6-diamino-purine bases, compounds useful for the preparation of such compounds, various uses thereof and methods for the determination of nucleic acids using said compounds in the field of diagnostics.

Claims

1. A method for the determination of the presence, absence or amount of a nucleic acid comprising:

providing a sample suspected to contain the nucleic acid,

providing a nucleic acid binding compound comprising a backbone, said backbone having attached one or more heterocyclic groups capable of base pairing to nucleobases, characterized in that at least one of said heterocyclic groups is a group of formula I,

2. The method according to claim 1, wherein the nucleic acid binding compound comprises a reporter group.

3. The method according to claim 2 wherein the reporter group is a fluorescent label.

4. The method according to claim 3 wherein the nucleic acid binding compound further comprises a quenching agent which quenches the fluorescence emission of the fluorescent label.

5. The method according to claim 4 wherein the fluorescent label is a fluorescein and wherein the quenching agent is a fluorescent rhodamine or cyanine.

6. The method according to claim 4, further comprising altering the spatial relationship between the fluorescent label and the quenching agent subsequent to hybridization.

7. The method according to claim 6, wherein alteration of the spatial relationship between the fluorescent label and the quenching agent is accomplished by exonuclease hydrolysis of the nucleic acid binding compound, wherein release of label occurs as a result of exonuclease hydrolysis.

8. The method according to claim 7, wherein the degree of hybridization between the nucleic acid binding compound and the nucleic acid is determined by the quantity of label that is released from the nucleic acid binding compound subsequent to hybridization.

9. The method according to claim 1, wherein the degree of hybridization between the nucleic acid binding compound and the nucleic acid is determined by the priming ability of the nucleic acid binding compound.

10. The method according to claim 9, wherein priming occurs as part of a polymerase chain reaction.

11. The method according to claim 2, wherein more than one nucleic acid binding compound is used.

12. The method according to claim 11, wherein two nucleic acid binding compounds are used.

13. The method according to claim 12, wherein the first of the two nucleic acid binding compounds comprises a fluorescence donor and the second of the two nucleic acid binding compounds comprises a fluorescence acceptor, and wherein the emission wavelengths of the fluorescence donor overlap the absorption wavelengths of the fluorescence acceptor.

14. The method according to claim 13 wherein the degree of hybridization is measured by the quantity of light transferred between the fluorescence donor and the fluorescence acceptor and emitted by the fluorescence acceptor.

15. The method according to claim 13 wherein the degree of hybridization is determined by the measurement of the melting temperature between the nucleic acid binding compound and the nucleic acid.

16. A method for detecting the presence of a target sequence in a nucleic acid, the method comprising

a) providing a nucleic acid which is to be tested for the presence of the target sequence;

b) providing a nucleic acid binding compound comprising a backbone, said backbone having attached one or more heterocyclic groups capable of base pairing to nucleobases, characterized in that at least one of said heterocyclic groups is a group of formula I,

17. The method according to claim 16, wherein multiple nucleic acids are tested for the presence of the target sequence, and wherein the nucleic acids have related target sequences.

18. The method according to claim 16, wherein the nucleic acid binding compound is a primer comprising an extendible 3′

-hydroxyl group.

19. The method according to claim 18, wherein the hybridized nucleic acids are identified by extending the primer with a polymerizing enzyme.

20. The method according to claim 18, wherein the nucleic acid binding compound is a primer in a polymerase chain reaction.