Dark quenchers for donor-acceptor energy transfer

• US 8,633,307 B2
• Filed: 04/22/2010
• Issued: 01/21/2014
• Est. Priority Date: 05/09/2000
• Status: Expired due to Fees

• 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:
  US12/765,844
• Publication Number:
  US 20110092679A1
• Time in Patent Office: 1,370 Days
• Field of Search: 536/23.1, 536/26.6, 530/350, 436/501
• US Class Current: 536/26.6
• 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 quencher-labeled conjugate, comprising a quencher moiety and a conjugated substance, wherein the quencher moiety comprises structure (Q):

2. The quencher-labeled conjugate of claim 1 in which the conjugated substance is a polynucleotide.

3. The quencher-labeled conjugate of claim 2 which further comprises a fluorescent moiety covalently attached to the polynucleotide, the fluorescence of which is capable of being quenched by the quencher moiety when the fluorescent and quencher moieties are in close proximity to one another.

4. The quencher-labeled conjugate of claim 3 in which the 3′

-terminus of the polynucleotide cannot be extended by a polymerase.

5. The quencher-labeled conjugate of claim 4 in which the quencher moiety is attached to the 3′

-terminal nucleotide of the polynucleotide and the fluorescent moiety is attached to the 5′

-terminal nucleotide of the polynucleotide.

6. The quencher-labeled conjugate of claim 4 in which the quencher moiety is attached to the 3′

-terminus of the polynucleotide.

7. The quencher-labeled conjugate of claim 4 in which the fluorescent moiety is attached to the 5′

-terminus of the polynucleotide.

8. The quencher-labeled conjugate of claim 4 in which the fluorescent moiety is attached to the 3′

-terminal nucleotide of the polynucleotide and the quencher moiety is attached to the 5′

-terminal nucleotide of the polynucleotide.

9. The quencher-labeled conjugate of claim 4 in which the fluorescent moiety is attached to the 3′

-terminus of the polynucleotide.

10. The quencher-labeled conjugate of claim 4 in which the quencher moiety is attached to the 5′

-terminus of the polynucleotide.

12. The quencher-labeled conjugate of claim 1 in which the quencher moiety (Q) comprises structure (Q1):

13. The quencher-labeled conjugate of claim 12 in which the electron-withdrawing groups are each, independently of one another, selected from NO₂, CN, CF₃, CO₂H, CO₂R′

, C(O)NH₂, N(O)NHR′

, C(O)NR′

R′

, CHO, C(O)R′

, SO₂R′

, SO₂CF₃, SO₂OR′

, SO₃H, NO and C₅-C₁₄ aryl, where R′

is H, C₁-C₁₂ alkyl or C₅-C₁₄ aryl.

14. The quencher-labeled conjugate of claim 12 in which R¹⁰ is NO₂.

15. The quencher-labeled conjugate of claim 12 in which at least one of R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ is OCH₃.

16. The quencher-labeled conjugate of claim 12 in which Q1 is selected from:

11. A quencher-labeled conjugate, comprising a quencher moiety and a conjugated substance, wherein the quencher moiety comprises structure (Q):