Dark quenchers for donor-acceptor energy transfer

• US 9,139,610 B2
• Filed: 10/29/2013
• Issued: 09/22/2015
• Est. Priority Date: 05/09/2000
• Status: Expired due to Term

• Current Assignee: HSB Corporate Trust Company (UK) Limited
• Original Assignee: Biosearch Technologies Incorporated
• Inventors: Cook, Ronald M., Lyttle, Matt, Dick, Daren
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/066,041
• Publication Number:
  US 20140187763A1
• Time in Patent Office: 693 Days
• Field of Search: 536/23.1, 536/24.3, 536/26.6
• US Class Current: 1/1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07C 245/08 with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
• CPC Class Codes: C07D 241/46 Phenazines
• CPC Class Codes: C07F 9/091 with hydroxyalkyl compounds with further substituents on alkyl
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• 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: C09B 31/043 Amino-benzenes
• CPC Class Codes: C09B 56/00 Azo dyes containing other chromophoric systems
• CPC Class Codes: C09B 56/12 Anthraquinone-azo dyes from diazotised aminoanthracene C09B29/0022, azo dyes containing hydroxyl groups acylated with polyfunctional anthraquinone derivatives C09B43/26
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The present invention provides a family of dark quenchers, termed Black Hole Quenchers (“BHQs”), that are efficient quenchers of excited state energy but which are themselves substantially non-fluorescent. Also provided are methods of using the BHQs, probes incorporating the BHQs and methods of using the probes.

Claims

1. A nucleic acid having linked thereto:

a quencher of excited state energy having a structure comprising at least three residues selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond; and

a minor groove binder.

2. The nucleic acid according to claim 1, wherein said quencher is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher precursor having a structure according to Formula (I)

3. The nucleic acid according to claim 2, wherein at least one of X, Y and Y′

is;

4. The nucleic acid according to claim 3, wherein at least one of X, Y and Y′

is;

5. The nucleic acid according to claim 4, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

6. The nucleic acid according to claim 1, wherein said quencher is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher precursor having a structure according to Formula (II)

7. The nucleic acid according to claim 6, wherein at least one of X and Y is:

8. The nucleic acid according to claim 7, wherein at least one of X and Y is:

9. The nucleic acid according to claim 8, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

10. The nucleic acid according to claim 6, wherein

R² and R³ are members independently selected from aryl and aryl substituted with a member selected from amino, amino derivatives, nitro, C₁-C₆ alkyl, C₁-C₆ alkoxy and combinations thereof;

R¹ includes a structure according to Formula III;

11. The nucleic acid according to claim 10, wherein at least one of X and Y is:

12. The nucleic acid according to claim 11, wherein at least one of X and Y is:

13. The nucleic acid according to claim 12, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

14. The nucleic acid according to claim 1, wherein said quencher is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher precursor having a structure according to Formula IV:

15. The nucleic acid according to claim 14, wherein at least one of X and Y is:

16. The nucleic acid according to claim 15, wherein at least one of X and Y is:

17. The nucleic acid according to claim 16, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

18. The nucleic acid according to claim 1, wherein said quencher is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher precursor having a structure according to Formula V:

19. The nucleic acid according to claim 18, wherein at least one of X and Y is:

20. The nucleic acid according to claim 19, wherein at least one of X and Y is:

21. The nucleic acid according to claim 20, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

22. The nucleic acid according to claim 1, wherein said quencher is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher precursor having a structure according to Formula VI:

23. The nucleic acid according to claim 1, wherein said quencher is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher precursor having a structure which is a member selected from:

24. The nucleic acid according to claim 23, wherein at least one of X⁵ and X⁶ is:

25. The nucleic acid according to claim 24, wherein at least one of X⁵ and X⁶ is:

26. The nucleic acid according to claim 25, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.