Stabilized nucleic acid dark quencher-fluorophore probes

• US 9,803,240 B2
• Filed: 03/17/2014
• Issued: 10/31/2017
• Est. Priority Date: 04/01/2008
• Status: Active Grant

• Current Assignee: HSB Corporate Trust Company (UK) Limited
• Original Assignee: Biosearch Technologies Incorporated
• Inventors: Cook, Ronald M., Lyttle, Matt
• Primary Examiner(s): Otton, Alicia L
• Application Number:
  US14/215,272
• Publication Number:
  US 20140194611A1
• Time in Patent Office: 1,324 Days
• Field of Search: None
• CPC Class Codes: C07D 219/04 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C09B 31/02 Disazo dyes
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

The present invention provides a new class of solids supports for synthesis of modified oligomers of nucleic acids, and nucleic acid probes that have a format expediently synthesized on the new supports. Exemplary solid supports include at least one quencher bound through a linker to the solid support. Various exemplary embodiments include a moiety that stabilizes a duplex, triplex or higher order aggregation (e.g., hybridization) of nucleic acids of which the oligomer of the invention is a component. Other components of the solid support include moieties that stabilize aggregations of nucleic acids, e.g., intercalators, minor groove binding moieties, bases modified with a stabilizing moiety (e.g., alkynyl moieties, and fluoroalkyl moieties), and conformational stabilizing moieties, such as those described in commonly owned U.S. Patent Application Publication No. 2007/0059752.

Claims

1. A nucleic acid oligomer comprising at least one base, having a formula selected from:

2. The nucleic acid oligomer according to claim 1, wherein said quencher is of a structure according to Formula V:

3. The nucleic acid oligomer according to claim 2, wherein v is 1.

4. The nucleic acid oligomer according to claims 2, wherein said linkage fragment is NH or NR^t, wherein R^t is selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

5. The nucleic acid oligomer according to claim 1, wherein said quencher is of a structure according to Formula VI:

6. The nucleic acid oligomer according to claim 5, wherein v is 1.

7. The nucleic acid oligomer according to claim 6, wherein said quencher has a structure selected from:

8. The nucleic acid oligomer according to claim 5, wherein said linkage fragment is NH or NR^t, wherein R^t is selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

9. The nucleic acid oligomer according to claim 1, wherein said quencher is of a structure according to Formula (II)

10. The nucleic acid oligomer according to claim 9, wherein said quencher is of a structure according to Formula (III):

11. The nucleic acid oligomer according to claim 9, wherein said quencher has the structure:

12. The nucleic acid oligomer according to claim 9, wherein said linkage fragment is NH or NR^t, wherein R^tis selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

13. The nucleic acid oligomer according to claim 1, wherein said nucleic acid oligomer is a probe.

14. The nucleic acid oligomer according to claim 13, wherein said probe is selected from molecular beacons, conformationally assisted probes, light up probes, and invader detection probes.

15. The nucleic acid oligomer according to claim 1, wherein said alkynyl moiety is selected from the group consisting of:

ethynyl, 1-propynyl, 1-butynyl, 1-pentynyl, 1,3-pentadiynyl, phenylethynyl, phenylethynyl, pyridine-ethynyl, pyrimidine-ethynyl, triazine-ethynyl, thiophene-ethynyl, thiazole-ethynyl, imidazole-ethynyl and substituted or unsubstituted C₁-C₁₀ alkynyl groups.

16. The nucleic acid oligomer according to claim 15, wherein the substituents for the C₁-C₁₀ alkynyl groups are selected from the group consisting of:

2-, 3-, and 4-pyridinyl;

triazine;

2-, 4- and 5-pyrimidinyl;

2-, 4- and 5-thiazolyl;

1-methyl-2-imidazolyl;

2- and 4-imidazolyl;

2-, 4- and 5-oxazolyl;

3-pyridinyl;

4-pyridinyl;

2-pyridinyl;

2- and 3-thienyl;

2- and 3-furanyl;

2- and 3-pyrrolyl;

propenyl (—

CH═

CH—

CH₃);

vinyl; and

—

C≡

C—

Z′

where Z′

is hydrogen (H) or C₁-C₁₀ alkyl, C₁-C₁₀ alkyl with 1 to 6 halogen atoms;

or C₁-C₁₀ heteroalkyl having 1 to 3 heteroatoms selected from the group consisting of O, N and S.

17. The nucleic acid oligomer according to claim 15, wherein said alkynyl moiety is 1-propynyl.

18. The nucleic acid oligomer according to claim 1, further comprising a fluorophore.

19. The nucleic acid oligomer according to claim 1 contains 8 to 30nucleomonomers.

20. A method for detecting or quantifying one or more target nucleic acid molecules, said method comprising:

(a) providing a sample;

(b) contacting the sample with a composition comprising the oligomer according to claim 1 under conditions that allow hybridization of said oligomer to said target nucleic acid molecules; and

(c) detecting or quantifying one or more target nucleic acid molecules of the sample that have hybridized to one or more said oligomer.