Dark quenchers for donor-acceptor energy transfer

• US 10,301,349 B2
• Filed: 03/30/2015
• Issued: 05/28/2019
• Est. Priority Date: 05/09/2000
• Status: Active Grant

• Current Assignee: Biosearch Technologies Incorporated
• Original Assignee: Biosearch Technologies Incorporated
• Inventors: Cook, Ronald M., Lyttle, Matt, Dick, Daren
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/673,118
• Publication Number:
  US 20150197792A1
• Time in Patent Office: 1,520 Days
• Field of Search: 536 266
• 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 probe comprising a nucleic acid having attached thereto:

a first quencher moiety 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 second quencher moiety of excited state energy.

2. The nucleic acid probe according to claim 1, wherein the second quencher moiety comprises 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.

3. The nucleic acid probe according to claim 1, wherein the second quencher moiety is covalently bonded to said nucleic acid at an internal position of the nucleic acid.

4. The nucleic acid probe according to claim 3, wherein said nucleic acid probe is in a format selected from molecular beacons, scorpion probes, sunrise probes, conformationally assisted probes, and TaqMan™

probes.

5. The nucleic acid probe according to claim 1, further comprising a fluorescent moiety attached to said nucleic acid.

6. The nucleic acid probe according to claim 5, wherein the fluorescent moiety is attached to a member selected from the 5′

-terminal nucleotide, the 3′

-terminal nucleotide of the probe, and a nucleotide internal to the 5′

-terminal nucleotide, and the 3′

-terminal nucleotide.

7. The nucleic acid probe according to claim 6, wherein the fluorescent moiety is selected from the group consisting of xanthene, fluorescein, coumarin, rhodamine, and cyanine.

8. The nucleic acid probe according to claim 1, wherein at least one of the first quencher moiety and the second quencher moiety has a structure according to Formula (VI):

9. The nucleic acid probe of claim 8, wherein the first quencher moiety and the second quencher moiety independently have a structure according to Formula (VI).

10. The nucleic acid probe of claim 8, wherein R⁵ and R⁶ are substituted or unsubstituted C₁-C₆ alkyl.

11. The nucleic acid probe of claim 8, wherein at least one of the first quencher moiety and the second quencher moiety has a structure according to the formula:

12. The nucleic acid probe of claim 8, wherein the second quencher moiety has a structure according to formula (VI).

13. The nucleic acid probe according to claim 1, wherein at least one of the first quencher moiety and the second quencher moiety is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher moiety precursor having a structure according to Formula (I):

14. The nucleic acid probe according to claim 13, wherein at least one of X, Y and Y′

is;

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

is;

16. The nucleic acid probe according to claim 14, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

17. The nucleic acid probe according to claim 1, wherein at, least one of the first quencher moiety and the second quencher moiety is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher moiety precursor having a structure according to Formula (II):

18. The nucleic acid probe according to claim 17, wherein at least one of X and Y is:

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

20. The nucleic acid probe according to claim 18, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

21. The nucleic acid probe according to claim 17, 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.

22. The nucleic acid probe according to claim 17, wherein R¹ includes a structure according to Formula III:

23. The nucleic acid probe according to claim 1, wherein at least one of the first quencher moiety and the second quencher moiety is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher moiety precursor having a structure according to Formula IV:

24. The nucleic acid probe according to claim 23, wherein at least one of X and Y is:

25. The nucleic acid probe according to claim 24, wherein at least one of X and Y is:

26. The nucleic acid probe according to claim 24, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

27. The nucleic acid probe according to claim 1, wherein at least one of the first quencher moiety and the second moiety is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher moiety precursor having a structure according to Formula V:

28. The nucleic acid probe according to claim 27, wherein at least one of X and Y is:

29. The nucleic acid probe according to claim 28, wherein at least one of X and Y is:

30. The nucleic acid probe according to claim 28, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

31. The nucleic acid probe according to claim 1, wherein at least one of the first quencher moiety and the second quencher moiety is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher moiety precursor having a structure according to Formula VI:

32. The nucleic acid probe according to claim 1, wherein at least one of the first quencher moiety and the second quencher moiety is covalently attached to said nucleic acid by reaction of a nucleic acid and a quencher moiety precursor having a structure which is a member selected from:

33. The nucleic acid probe according to claim 32, wherein at least one of X⁵ and X⁶ is:

34. The nucleic acid probe according to claim 33, wherein at least one of X⁵ and X⁶ is:

35. The nucleic acid probe according to claim 33, wherein X³ is cyanoethyl;

and X⁴ is isopropyl.

36. A method for detecting a nucleic acid target sequence, said method comprising:

(a) contacting said target sequence with a detector nucleic acid, wherein the detector nucleic acid is the nucleic acid probe of claim 5, wherein said detector nucleic acid comprises a single-stranded target binding sequence,

wherein said detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between said fluorescent moiety and at least one of said first quencher moiety and said second quencher moiety when said fluorescent moiety is excited;

(b) hybridizing said target binding sequence to said target sequence, thereby altering said conformation of said detector nucleic acid, causing a change in a fluorescence parameter; and

(c) detecting said change in said fluorescence parameter, thereby detecting said nucleic acid target sequence.

37. The method according to claim 36, wherein said target sequence is amplified by a method selected from Strand Displacement Amplification, Polymerase Chain Reaction, 3SR, TMA, and NASBA.

38. The method according to claim 36, wherein said change in said fluorescence parameter is detected as an indication of the presence of said target sequence.

39. The method according to claim 36, wherein said fluorescence parameter is detected in real-time.

40. The method according to claim 36, wherein said detector nucleic acid has an intramolecularly base-paired secondary structure which comprises a portion of said target binding sequence.

41. The method according to claim 36, wherein said nucleic acid target sequence is in a mixture.

42. A method of ascertaining whether a first nucleic acid and a second nucleic acid hybridize, said first nucleic acid being a nucleic acid probe according to claim 1, said method comprising:

(a) contacting said first nucleic acid with said second nucleic acid;

(b) detecting an alteration in a fluorescent property of a member selected from said first nucleic acid, said second nucleic acid and a combination thereof, thereby ascertaining whether the hybridization occurs.

43. A method of ascertaining whether a first nucleic acid and a second nucleic acid hybridize, said method comprising:

(a) contacting the first nucleic acid with the second nucleic acid, wherein the first nucleic acid is the nucleic acid probe of claim 1;

(b) detecting an alteration in a fluorescent property of the first nucleic acid, thereby ascertaining whether the hybridization occurs.

44. A method of detecting a nucleic acid target sequence, said method comprising:

(a) amplifying the nucleic acid target sequence in a TaqMan™

assay in the presence of a nucleic acid probe of claim 1, wherein hybridization of the nucleic acid probe to the amplification product and/or cleavage of the nucleic acid probe after its hybridization to the amplification product results in a change in a fluorescence parameter;

(b) detecting said change in said fluorescence parameter, thereby detecting said nucleic acid target sequence.

45. A mixture comprising a nucleic acid probe according to claim 1.

46. A multiplexing nucleic acid probe mixture comprising first and second nucleic acid probes according to claim 1, wherein the nucleic acid of the first nucleic acid probe and the nucleic acid of the second nucleic acid probe are different.

47. The multiplexing nucleic acid probe mixture according to claim 46, wherein the nucleic acid sequence of the first nucleic acid probe and the nucleic acid sequence of the second nucleic acid probe are different.

48. A multiplexing nucleic acid probe mixture comprising first and second nucleic acid probes according to claim 1, wherein said first nucleic acid probe has attached thereto a first fluorescent moiety, and said second nucleic acid probe has attached thereto a second fluorescent moiety, wherein said first fluorescent moiety and said second fluorescent moiety are different fluorescent moieties.

49. A multiplexing nucleic acid probe mixture comprising first and second nucleic acid probes according to claim 1, wherein at least one of:

(i) the first quencher moiety of excited state energy of the first nucleic acid probe and the first quencher moiety of excited state energy of the second nucleic acid probe; and

(ii) the second quencher moiety of excited state energy of the first nucleic acid probe and the second quencher moiety of excited state energy of the second nucleic acid probe,

are different quencher of excited state energy moieties.