Dark Quenchers For Donor-Acceptor Energy Transfer

• US 20110092679A1
• Filed: 04/22/2010
• Published: 04/21/2011
• Est. Priority Date: 05/09/2000
• Status: Active Grant

• Current Assignee: HSB Corporate Trust Company (UK) Limited
• Original Assignee: Biosearch Technologies Incorporated
• Inventors: Lyttle, Matt, Cook, Ronald M., Dick, Daren
• Granted Patent: US 8,633,307 B2
• Time in Patent Office: Days
• US Class Current: 530/409
• 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 of excited state energy having a structure comprising at least three radicals selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and combinations thereof, wherein at least two of said radicals are covalently linked via an exocyclic diazo bond, said quencher further comprising a reactive functional group providing a locus for conjugation of said quencher to a carrier molecule.

2. The quencher according to claim 1, wherein at least two of said radicals are members selected from aryl and substituted aryl groups.

3. The quencher according to claim 1, wherein one of said radicals is a member selected from substituted or unsubstituted polycyclic aryl and substituted or unsubstituted polycyclic heteroaryl groups.

4. The quencher according to claim 1, wherein said quencher is attached to a carrier molecule.

5. The quencher according to claim 4, wherein said carrier molecule is a member selected from peptides, saccharides, nucleic acids, proteins, antibodies and combinations thereof.

6. The quencher according to claim 5, wherein said carrier molecule further comprises a fluorophore.

7. The quencher according to claim 6, wherein said fluorophore and said quencher comprise a donor-acceptor energy transfer pair.

8. The quencher according to claim 6, wherein said carrier molecule is a nucleic acid.

9. The quencher according to claim 8, wherein said quencher is covalently attached to a member selected from the 3′

-terminus and the 5′

-terminus of said nucleic acid.

10. The quencher according to claim 8, wherein said quencher is covalently attached to said nucleic acid at a position between the 3′

-terminus and the 5′

-terminus of said nucleic acid.

11. The quencher according to claim 8, wherein said nucleic acid is a member selected from molecular beacons, scorpion probes, sunrise probes, conformationally assisted probes and TaqMan™

probes.

12. The quencher according to claim 6, wherein said carrier molecule is a peptide comprising a cleavage recognition site for an enzyme.

13. The quencher according to claim 12, wherein said peptide comprises a cleavage recognition site for a protease.

14. The quencher according to claim 12, wherein said cleavage recognition site is for an enzyme selected from trypsin, enterokinase, HIV-1 protease, prohormone convertase, interleukin-1b-converting enzyme, adenovirus endopeptidase, cytomegalovirus assemblin, leishmanolysin, β

-secretase for amyloid precursor protein, thrombin, renin, angiotensin-converting enzyme, cathepsin-D and a kininogenase.

15. The quencher according to claim 12, wherein said peptide is of a length and conformation that allows fluorescent energy transfer between said fluorophore and said quencher.

16. The quencher according to claim 12, wherein said peptide is a peptide-nucleic acid.

17. The quencher according to claim 4, wherein said quencher is attached to a carrier molecule comprising a solid support.

18. The quencher according to claim 17, wherein said quencher is attached to a nucleic acid attached to said solid support.

19. The quencher according to claim 18, further comprising a linker group between said nucleic acid and said solid support.

20. The quencher according to claim 17, further comprising a linker group between said quencher and said solid support

21. The quencher according to claim 1, wherein said quencher has substantially no native fluorescence.

22. The quencher according to claim 1, wherein said quencher has an absorbance maximum of from about 400 nm to about 760 nm.

23. The quencher according to claim 22, wherein said quencher has an absorbance maximum of from about 500 nm to about 600 nm.

24. A quencher of excited state energy having the formula:

25. The compound according to claim 24 wherein at least one member selected from the group consisting of m, n and f is at least 1.

26. The quencher according to claim 24, wherein at least one of X, Y and Y′

is;

27. The quencher according to claim 26, wherein a member selected from R⁹, R¹⁰ and combinations thereof comprises a polyether.

28. The quencher according to claim 27, wherein said polyether is a member selected from poly(ethylene glycol), poly(propyleneglycol) and copolymers thereof.

29. The quencher according to claim 26, wherein X is:

30. The quencher according to claim 29, wherein

X³ is cyanoethyl;

and

X⁴ is isopropyl.

31. The quencher according to claim 29, wherein p and q are numbers independently selected from 2 to 16, inclusive.

32. The quencher according to claim 24, wherein R¹ and R² are members independently selected from aryl substituted with a member selected from the group consisting of —

NR′

R″

, nitro, substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₁-C₆ alkoxy and combinations thereof,

wherein R′ and

R″

are members independently selected from H, and substituted or unsubstituted C₁-C₆ alkyl.

33. The quencher according to claim 24, wherein at least one member selected from the group consisting of R¹, R² and R³ comprises:

34. A quencher of excited state energy having the formula:

35. The compound according to claim 34, wherein

X is —

NR′

R″

, wherein R′ and

R″

are members independently selected from H, and alkyl or substituted alkyl groups;

m is 1;

n is 0;

s is 1; and

R¹, R² and R³ are members independently selected from substituted and unsubstituted aryl.

36. The quencher according to claim 34, wherein X is:

37. The quencher according to claim 36, wherein a member selected from R⁹, R¹⁰ and combinations thereof comprises a polyether.

38. The quencher according to claim 37, wherein said polyether is a member selected from poly(ethylene glycol), poly(propyleneglycol) and copolymers thereof.

39. The quencher according to claim 36, wherein X is:

40. The quencher according to claim 39, wherein

X³ is cyanoethyl;

and

X⁴ is isopropyl.

41. The quencher according to claim 39, wherein p and q are numbers independently selected from 2 to 16, inclusive.

42. The quencher according to claim 35, wherein R¹ and R² are members independently selected from aryl substituted with a member selected from the group consisting of —

NR′

R″

, nitro, substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₁-C₆ alkoxy and combinations thereof

wherein R′ and

R″

are members independently selected from H, and substituted or unsubstituted C₁-C₆ alkyl.

43. A quencher of excited state energy having the structure:

44. The compound according to claim 43, wherein

X is —

NR′

R″

, wherein R′ and

R″

are members independently selected from H, and alkyl or substituted alkyl groups;

m is 1; and

R¹, R² and R³ are members independently selected from substituted and unsubstituted aryl.

45. The quencher according to claim 44, wherein X is:

46. The quencher according to claim 45, wherein a member selected from R⁹, R¹⁰ and combinations thereof comprises a polyether.

47. The quencher according to claim 46, wherein said polyether is a member selected from poly(ethylene glycol), poly(propyleneglycol) and copolymers thereof.

48. The quencher according to claim 44, wherein X is:

49. The quencher according to claim 48, wherein

X³ is cyanoethyl;

and

X⁴ is isopropyl.

50. The quencher according to claim 48, wherein p and q are numbers independently selected from 2 to 16, inclusive.

51. The quencher according to claim 44, wherein R¹, R² and R³ are members independently selected from the group consisting of aryl substituted with a member selected from —

NR′

R″

, nitro, substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₁-C₆ alkoxy and combinations thereof,

wherein R′ and

R″

are members independently selected from H, and substituted or unsubstituted C₁-C₆ alkyl.

52. A quencher of excited state energy having the structure:

53. A quencher of excited state energy according to claim 52 having the structure:

54. The quencher according to claim 53, wherein

X³ is cyanoethyl;

and

X⁴ is isopropyl.

55. The quencher according to claim 53, wherein p and q are independently selected numbers between 2 and 16, inclusive.

56. A quencher of excited state energy having a structure which is a member selected from:

57. A method for determining whether a sample contains an enzyme, said method comprising:

(a) contacting said sample with a peptide construct comprising

i) a fluorophore;

ii) a quencher comprising at least three residues selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond; and

iii) a cleavage recognition site for said enzyme,

 

wherein said peptide is in a conformation allowing donor-acceptor energy transfer between said fluorophore and said quencher when said fluorophore is excited;

(b) exciting said fluorophore; and

(c) determining a fluorescence property of said sample, wherein the presence of said enzyme in said sample results in a change in said fluorescence property.

58. A method for determining whether a compound alters an activity of an enzyme, said method comprising:

(a) contacting a sample comprising said enzyme and said compound with a peptide construct comprising

i) a fluorophore;

ii) a quencher comprising at least three residues selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond; and

iii) a cleavage recognition site for said enzyme,

 

wherein said peptide is in a conformation allowing donor-acceptor energy transfer between said fluorophore and said quencher when said fluorophore is excited;

(b) exciting said fluorophore; and

(c) determining a fluorescence property of said sample, wherein said activity of said enzyme in said sample results in a change in said fluorescence property.

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

(a) contacting said target sequence with a detector oligonucleotide comprising a single-stranded target binding sequence, said detector oligonucleotide having linked thereto,

i) a fluorophore; and

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

 

wherein said detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between said fluorophore and said quencher when said fluorophore is excited;

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

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

60. The method according to claim 59, wherein said detector oligonucleotide has a format selected from molecular beacons, scorpion probes, sunrise probes, conformationally assisted probes and TaqMan™

probes.

61. The method according to claim 59, wherein said intramolecularly associated secondary structure is a member selected from hairpins, stem-loop structures, pseudoknots, triple helices and conformationally assisted structures.

62. The method according to claim 59, wherein said complementary strand is synthesized in a target amplification reaction.

63. The method according to claim 59, wherein said complementary strand is synthesized by extension of the target sequence using said detector oligonucleotide as a template.

64. The method according to claim 59, wherein the intramolecularly associated secondary structure comprises a totally or partially single-stranded endonuclease recognition site.

65. The method according to claim 59, wherein said change in fluorescence is detected as an indication of the presence of said target sequence.

66. The method according to claim 59, wherein said fluorescence parameter is detected in-real time.

67. The method according to claim 59, wherein said intramolecularly base-paired secondary structure comprises a portion of said target binding sequence.

68. A method for detecting amplification of a target sequence comprising, in an amplification reaction:

(a) hybridizing to said target sequence a detector oligonucleotide comprising a single-stranded target binding sequence and an intramolecularly associated secondary structure 5′

to said target binding sequence, wherein at least a portion of said detector sequence forms a single stranded tail which is available for hybridization to said target sequence, said detector oligonucleotide having linked thereto,

i) a fluorophore; and

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

 

wherein said detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between said fluorophore and said quencher when said fluorophore is excited;

(b) extending said hybridized detector oligonucleotide on said target sequence with a polymerase to produce a detector oligonucleotide extension product and separating said detector oligonucleotide extension product from said target sequence;

(c) hybridizing a primer to said detector oligonucleotide extension product and extending the primer with said polymerase, thereby linearizing said intramolecularly associated secondary structure and producing a change in a fluorescence parameter; and

(d) detecting said change in said fluorescence parameter, thereby detecting said target sequence.

69. The method according to claim 68, wherein said target sequence is amplified by a method selected from Strand Displacement Amplification, Polymerase Chain reaction 3SR, TMA and NASBA.

70. The method according to claim 68, wherein said secondary structure further comprises a partially or entirely single-stranded restriction endonuclease site.

71. The method according to claim 68, wherein a change in fluorescence intensity is detected.

72. The method according to claim 71, wherein said change in fluorescence intensity is detected in real-time.

73. The method according to claim 68, wherein said intramolecularly base-paired secondary structure comprises a portion of said target binding sequence.

74. A method of ascertaining whether a first nucleic acid and a second nucleic acid hybridize, said first nucleic acid comprising a quencher of excited state energy having a structure comprising at least three residues selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond, covalently linked to said nucleic acid and a second nucleic acid, 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 said hybridization occurs.

75. The method according to claim 74, wherein said second nucleic acid comprises a fluorophore covalently attached thereto.

76. A microarray comprising a quencher of excited state energy having a structure comprising at least three residues selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond, said quencher being conjugated directly to a solid support or to a carrier molecule attached to said solid support.

77. The microarray according to claim 76, wherein said carrier molecule is a member selected from a nucleic acid, a peptide, a peptide nucleic acid, a small molecule bioactive species and combinations thereof.

78. The microarray according to claim 76, wherein said solid support is divided into a first region and a second region, said first region having attached thereto a first said quencher of excited state energy attached to a first carrier molecule and said second region having attached thereto a second said quencher of excited state energy attached to a second carrier molecule.

79. The microarray according to claim 78, wherein said first and second carrier molecules are members independently selected from nucleic acids, peptides and peptide nucleic acids.

80. The microarray according to claim 78, wherein said first said quencher of excited state energy and said second quencher of excited state energy have different structures.

81. A method of probing a microarray for the presence of a compound, said method comprising:

(a) contacting said microarray with a probe interacting with said compound, said probe comprising a quencher of excited state energy having a structure comprising at least three residues selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond;

(b) detecting a difference in a fluorescence property of a member selected from said probe, said compound and combinations thereof, thereby ascertaining the presence of said compound.

82. The method according to claim 81, wherein said compound is a member selected from nucleic acids, peptide, peptide nucleic acids and combinations thereof.

83. A mixture comprising at least a first carrier molecule and a second carrier molecule,

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

and

said second carrier molecule has covalently bound thereto a second quencher of excited state energy having a structure comprising at least three residues selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond.

84. The mixture according to claim 83, wherein said first carrier molecule and said second carrier molecule are different molecules.

85. The mixture according to claim 83, wherein said first quencher of excited state energy and said second quencher of excited state energy are different quenchers.

86. The mixture according to claim 83, wherein at least one of said first carrier molecule and said second carrier molecule further comprise a fluorophore covalently bound thereto.

87. A method for detecting or quantifying a molecular species, said method comprising:

(a) contacting said species with a mixture according to claim 83; and

(b) detecting a change in a fluorescent property of a component of said mixture, said molecular species and a combination thereof, thereby detecting or quantifying said molecular species.