Nucleic acid detection with energy transfer

• US 5,827,653 A
• Filed: 05/20/1996
• Issued: 10/27/1998
• Est. Priority Date: 09/23/1993
• Status: Expired due to Fees

• Current Assignee: Astrazeneca UK Limited (Astrazeneca PLC)
• Original Assignee: Zeneca Incorporated (Astrazeneca PLC)
• Inventors: Sammes, Peter George, Garman, Andrew John
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/619,724
• Time in Patent Office: 890 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 435/183, 436/164, 436/73, 436/94, 436/172, 436/800, 436/805, 252/700, 536/24.3, 536/26.6, 935/8, 935/76, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C07D 471/04 Ortho-condensed systems
• 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: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: G01N 2458/40 Rare earth chelates
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

A method for the detection of a nucleic acid analyte by complementary probe hybridisation and formation of a chelated lanthanide complex which, upon irradiation by light, results in a characteristic delayed luminescence emission.

Claims

1. A method for detecting a nucleic acid analyte in a sample, which method comprises contacting the sample with (i) a binding entity complementary to the analyte and to which is attached a first partner of a ligand to metal energy transfer system, (ii) a lanthanide ion, and (iii) a second partner of the system to which is attached a duplex binder;

wherein the first partner of the ligand to metal energy transfer system is a complexing sensitiser and the second partner is a chelating agent, or vice versa;

such that upon complementary binding of the analyte and the binding entity to form a nucleic acid duplex, interaction of the duplex binder with the duplex occurs and allows the first and the second partners of the system to form a closed chelated system around the lanthanide ion and, upon irradiation, emission of light from the lanthanide ion indicates the presence of the analyte in the sample.

2. A method as claimed in claim 1 wherein the duplex binder is selected from the group consisting of an intercalator and a groove binder.

3. A method as claimed in claim 1 wherein the binding entity is a polynucleotide to which the first or the second partner is attached.

4. A method as claimed in claim 3 wherein the binding entity comprises a blocked terminal nucleotide.

5. A method as claimed in any one of the previous claims wherein the binding entity comprises one or more mismatched bases opposite a complementary analyte nucleic acid sequence.

6. A method as claimed in claim 5 wherein a position of the one or more mismatched bases is selected such that duplex binding and hence formation of the closed chelated system around the lanthanide ion are modulated.

7. A method as claimed in claim 1 wherein the lanthanide ion is an europium ion.

8. A method as claimed in claim 1 wherein the complexing sensitiser comprises an aromatic group.

9. A method as claimed in claim 1 wherein the complexing sensitiser comprises a heterocyclic aromatic group.

10. A method as claimed in claim 1 wherein the complexing sensitiser comprises an optionally substituted dipyridyl or phenanthroline group.

11. A method as claimed in claim 1 wherein the complexing sensitiser comprises an optionally substituted phenanthroline-2,6-dicarboxylic acid group.

12. A method as claimed in claim 1 wherein the chelating agent is chelated with the lanthanide ion before addition to the assay medium.

13. A method as claimed in claim 1 wherein the binding entity is a polynucleotide to which a chelated lanthanide ion is attached.

14. A method as claimed in claim 1 wherein one partner of the ligand to metal energy transfer system is a complexing sensitiser linked to a duplex binder.

15. A method as claimed in claim 1 wherein the binding entity is an allele-specific polynucleotide.

16. A method as claimed in claim 1 which comprises amplification of a target sequence to provide the nucleic acid analyte.

17. A method as claimed in claim 16 wherein one or more components of the method are present during amplification of the target sequence.

18. A method as claimed in claim 1 wherein the binding entity is comprised of two or more polynucleotide sequences.

19. A method as claimed in claim 18 wherein the first and the second partners of the ligand to metal energy transfer system are attached to different polynucleotide sequences.

20. A method as claimed in claim 1 wherein the chelating agent is a polyfunctional compound comprising carboxylic acid, amide and/or ether moieties.

21. A method as claimed in claim 20 wherein the chelating agent is a derivative of a compound selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and trans-1,2-diaminocyclohexanetetraacetic acid (DCTA).

22. A method as claimed in claim 1 wherein a Forster energy transfer acceptor is used to receive energy from the closed chelated system around the lanthanide ion and to emit light.

23. A method as claimed in claim 22 wherein the Forster energy transfer acceptor is bound to a polynucleotide.

24. A method as claimed in claim 1 for detection of more than one nucleic acid analyte in a sample.

25. A method as claimed in claim 1 wherein the characteristics of light emission are measured to indicate the nature and/or quantity of the nucleic acid analyte.

26. A method as claimed in claim 1 which is a homogeneous assay.

27. An assay kit which comprises (i) a binding entity complementary to the analyte and to which is attached a first partner of a ligand to metal energy transfer system, (ii) a lanthanide ion, (iii) a second partner of the system to which is attached a duplex binder;

wherein the first partner of the ligand to metal energy transfer system is a complexing sensitiser and the second partner is a chelating agent, or vice versa; and

further comprising one or more of the following;

buffer(s), amplification primers and instructions for use of the kit.

28. An assay kit as claimed in claim 27 wherein the amplification primers are for allele specific amplification.

33. A compound as claimed in any one of claims 27 and 29-31 wherein the duplex binder is selected from the group consisting of an intercalator and a groove binder.

29. A compound which comprises a duplex binder covalently linked to a lanthanide ion sensitiser.

30. A compound as claimed in claim 29 which comprises a phenanthroline-2,6-dicarboxylic acid group linked to a phenanthridine group.

31. A compound as claimed in claim 30 of the formula ##STR3##

32. A composition which comprises a duplex binder covalently linked to a lanthanide ion chelating agent and a lanthanide ion.