Fluorescent quenching detection reagents and methods

• US 20040191796A1
• Filed: 06/25/2003
• Published: 09/30/2004
• Est. Priority Date: 12/08/1999
• Status: Abandoned Application

• Current Assignee: Epoch Pharmaceuticals, Inc.
• Original Assignee: Epoch Pharmaceuticals, Inc.
• Inventors: Gall, Alexander A., Dempcy, Robert O., Lukhtanov, Eugeny Alexander, Reed, Michael W.
• Application Number:
  US10/606,644
• Publication Number:
  US 20040191796A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/572 Five-membered rings
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• 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 29/0813 substituted by OH, O-C(=X)-R, O-C(=X)-X-R, O-R (X being O,S,NR; R being hydrocarbonyl)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Oligonucleotide-fluorophore-quencher conjugates wherein the fluorophore moiety has emission wavelengths in the range of about (300) to about (800) nm, and or where the quencher includes a substituted 4-(phenyldiazenyl)phenylamine structure provide improved signal to noise ratios and other advantageous characteristics in hybridization and related assays. The oligonucleotide-fluorophore-quencher conjugates can be synthesized by utilizing novel phosphoramidite reagents that incorporate the quencher moiety based on the substituted 4-(phenyldiazenyl)phenylamine structure, and or novel phosphoramidite reagents that incorporate a fluorophore moiety based on the substituted coumarin, substituted 7-hydroxy-3H-phenoxazin-3-one, or substituted 5,10-dihydro-10 [phenyl]pyrido[2,3-d;6,5-d′]dipyrimidine-2,4,6,8-(1H, 3H, 7H, 9H, 10H)-tetrone structure. Oligonucleotide-fluorophore-quencher-minor groove binder conjugates including a pyrrolo[4,5-e]indolin-7-yl}carbonyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indolin-7-caroxylate (DPI₃) moiety as the minor groove binder and the substituted 4-(phenyldiazenyl)phenylamine moiety as the quencher, were synthesized and have substantially improved hybridization and signal to noise ratio properties.

Claims

1. An oligonucleotide conjugate having the formula

FL-ODN-Q

where ODN is an oligonucleotide or nucleic acid;

FL is a fluorophore moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and

Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —

H, halogen, —

O(CH₂)_nCH₃, —

(CH₂)_nCH₃ where n=0 to 5, —

NO₂, —

SO₃, —

N[(CH₂)_n′

,CH₃]₂ where n′

=0 to 5 or —

CN, and R₅=—

H or —

(CH₂)_n′

CH₃ where n″

=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.

2. An oligonucleotide conjugate in accordance with claim 1 where R₀ is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is H or Cl in the 2 position of the benzene nucleus, and R₃ and R₄ are hydrogen and R₅ is ethyl.

3. An oligonucleotide conjugate in accordance with claim 1 where the quencher moiety and the linker attaching it to the ODN comprises the structures selected from the moieties shown by the formulas Q-1, Q-2 and Q-3

4. An oligonucleotide conjugate in accordance with claim 3 further comprising a minor groove binder moiety attached to the quencher-linker conjugate through one of the valence bonds designated a or b.

5. An oligonucleotide conjugate in accordance with claim 1 where the quencher moiety and of the linker attaching it to the ODN comprises the structures selected from the moieties shown by the formulas Q-4, and Q-5

6. A phosphoramidite reagent for preparing an oligonucleotide-fluorophore-quencher conjugate, the reagent including the moiety

7. A phosphoramidite reagent in accordance with claim 6 having the formula selected from the group consisting of the formulas designated PA-1, PA-2 and PA-3

8. A phosphoramidite reagent in accordance with claim 7 that has the formula designated PA-1.

9. A phosphoramidite reagent in accordance with claim 7 that has the formula designated PA-2.

10. A phosphoramidite reagent in accordance with claim 7 that has the formula designated PA-3.

11. A phosphoramidite reagent in accordance with claim 7 where R_o is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2 position of the benzene nucleus, and R₃ and R₄ are hydrogen and R₅ is ethyl.

12. A covalently linked solid support and quencher conjugate suitable for oligonucleotide synthesis, having the structure

13. A covalently linked solid support and quencher conjugate in accordance with claim 12 selected from the structures

14. A covalently linked solid support and quencher conjugate in accordance with claim 13 where R_ois H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2 position of the benzene nucleus, and R₅ is ethyl.

15. An oligonucleotide conjugate having the formula

FL-ODN-Q-MGB

where ODN is an oligonucleotide or nucleic acid;

FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and

Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —

H, halogen,—

O(CH2)_nCH₃, —

(CH₂)_nCH₃ where n=0 to 5, —

NO₂, —

SO₃, —

N[(CH₂)_n′

CH₃]₂ where n′

=0 to 5 or —

CN, and R₅=—

H, —

(CH₂)_n″

CH₃ or —

(CH₂)_n″

—

where n″

=0 to 5, and

MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms.

16. An oligonucleotide conjugate in accordance with claim 15 where the MGB moiety is attached to the quencher moiety, and the covalently bonded MGB -Q moiety has the structure

17. An oligonucleotide conjugate in accordance with claim 16 where R_o is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is H or Cl in the 2 position of the benzene nucleus, and R₃ and R₄ are hydrogen.

18. A covalently bonded minor groove binder and quencher reagent for oligonucleotide synthesis, having the formula

19. A covalently bonded minor groove binder and quencher reagent in accordance with claim 18 wherein X₃ is pentafluorophenyloxy.

20. A covalently bonded minor groove binder and quencher reagent in accordance with claim 18 wherein X₃ is NH-LINKER-CPG or O-LINKER-CPG.

21. A covalently bonded minor groove binder and quencher reagent in accordance with claim 18 where R_o is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is H or Cl in the 2 position of the benzene nucleus, R₃ and R₄ are hydrogen and v=t=3.

22. An oligonucleotide conjugate having the formula

FL-ODN-Q

where ODN is an oligonucleotide or nucleic acid;

Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and

FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, said fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,

wherein R₈and R₉ independently are H, halogen, —

NO₂, —

SO₃, —

C(═

O)NH₂, or —

CN;

—

OR_nn, —

SR_nn, —

OR_nn, —

NHR_nn,—

N[R_nn]2 where R_nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;

R₁₀, and R₁₁ independently are H, —

CN , —

OR₁₂, —

N(R₁₂)₂, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃, —

NO₂, —

SO₃, —

C(═

O)NH₂, —

N[(CH₂)_nCH₃]₂ where n=0 to 5, or R₁₂ is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons;

R₁₅ is H or alkyl of 1 to 10 carbons;

R₁₆ is alkyl of 1 to 10 carbons, and

the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.

23. An oligonucleotide conjugate in accordance with claim 22 where the fluorophore has the formula designated FL-1.

24. An oligonucleotide conjugate in accordance with claim 23 where R₈ is OC(O)CH(CH₃)₂ and R₉ is H.

25. An oligonucleotide conjugate in accordance with claim 22 where the fluorophore has the formula designated FL-2.

26. An oligonucleotide conjugate in accordance with claim 25 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁ is H.

27. An oligonucleotide conjugate in accordance with claim 22 where the fluorophore has the formula designated FL-3.

29. An oligonucleotide conjugate in accordance with claim 22 where the quencher moiety comprises the structure

30. An oligonucleotide conjugate in accordance with claim 22 comprising an additional minor groove binder moiety (MGB) attached to the quencher moiety through a linker having the length of 0 to approximately 30 atoms, whereby the oligonucleotide conjugate has the formula

FL-ODN-Q-MGB.

28. An oligonucleotide conjugate in accordance with claim 28 where R₁₅ is methyl and R₁₆ is n-propyl.

31. An oligonucleotide conjugate of the formula

32. An oligonucleotide conjugate in accordance with claim 31 where R_o is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is H or Cl in the 2 position of the benzene nucleus, and R₃ and R₄ are hydrogen.

33. An oligonucleotide conjugate in accordance with claim 31 where said fluorophore moiety has the structure selected from the group designated FL-1, FL-2 and FL-3,

34. A phosphoramidite reagent for preparing an oligonucleotide-fluorophore-quencher conjugate, the reagent selected from the group consisting of the structures designated PA-4, PA-5 and PA-6,

35. A phosphoramidite reagent in accordance with claim 34 that has the formula designated PA-4.

36. A phosphoramidite reagent in accordance with claim 35 where R₈ is —

OC(O)CH(CH₃)₂, R₉ is H, j is 2 and k is 6.

37. A phosphoramidite reagent in accordance with claim 34 that has the formula designated PA-5.

38. A phosphoramidite reagent in accordance with claim 37 where R₁₀ is OC(O)CH(CH₃)₂, R₁₁ is H and h is 3.

39. A phosphoramidite reagent in accordance with claim 34 that has the formula designated PA-6.

40. A phosphoramidite reagent in accordance with claim 39 where R₁₅ is methyl and R₁₆ is n-propyl.

41. A phosphoramidite reagent for preparing an oligonucleotide-fluorophore-quencher conjugate, the reagent having the formula

42. A phosphoramidite reagent in accordance with claim 41 where R_o is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2 position of the benzene nucleus, and R₃ and R₄ are hydrogen, R₅ is ethyl, n is 1 and q is 2.

43. An oligonucleotide conjugate having the formula

FL-ODN-MGB

where ODN is an oligonucleotide or nucleic acid;

MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms;

FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, said fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —

NO₂, —

SO₃, —

C(═

O)NH₂, or —

CN;

—

OR_nn, —

SR_nn, —

OR_nn,—

NHR<sub>nn,—

N[Rnn</sub>]₂ where R_nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;

R₁₀ and R₁₁ independently are H, —

CN, —

OR₁₂, —

N(R₁₂)₂, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃, —

NO₂, —

SO₃, —

C(═

O)NH₂, —

N[(CH₂)_nCH₃]₂ where n=0 to 5, or R₁₂ is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,

R₁₅ is H or alkyl of 1 to 10 carbons;

R₁₆ is alkyl of 1 to 10 carbons, and

the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.

44. An oligonucleotide conjugate in accordance with claim 43 where the fluorophore has the formula designated FL-1.

45. An oligonucleotide conjugate in accordance with claim 44 where R₈ is —

OC(O)CH(CH₃)₂ and R₉ is H.

46. An oligonucleotide conjugate in accordance with claim 43 where the fluorophore has the formula designated FL-2.

47. An oligonucleotide conjugate in accordance with claim 46 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁ is H.

48. An oligonucleotide conjugate in accordance with claim 43 where the fluorophore has the formula designated FL-3.

49. An oligonucleotide conjugate in accordance with claim 49where R₁₅ is methyl and R₁₆ is n-propyl.

50. An oligonucleotide conjugate having the formula

FL-ODN

where ODN is an oligonucleotide or nucleic acid;

FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, said fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —

NO₂, —

SO₃, —

C(═

O)NH₂, or —

CN;

—

OR_nn, —

SR_nn, —

OR_nn, —

NHR_nn,—

N[R_nn]₂ where R_nn, is independently H, an alkyl group of 1 to 10 carbons or an- alkanoyl group of 1 to 10 carbons;

R₁₀ and R₁₁ independently are H, —

CN, —

OR₁₂, —

N(R₁₂)₂, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃, —

NO₂, —

SO₃, —

C(=O)NH₂, —

N[(CH₂)_nCH₃]₂ where n=0 to 5,or R₁₂ is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,;

R₁₅ is H or alkyl of 1 to 10 carbons;

R₁₆ is alkyl of 1 to 10 carbons, and

the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.

51. An oligonucleotide conjugate in accordance with claim 50 where the fluorophore has the formula designated FL-1.

52. An oligonucleotide conjugate in accordance with claim 51 where R₈ is OC(O)CH(CH₃)₂ and R₉ is H.

53. An oligonucleotide conjugate in accordance with claim 50 where the fluorophore has the formula designated FL-2.

54. An oligonucleotide conjugate in accordance with claim 53 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁ is H.

55. An oligonucleotide conjugate in accordance with claim 50 where the fluorophore has the formula designated FL-3.

56. An oligonucleotide conjugate in accordance with claim 55 where R₁₅ is methyl and R₁₆ is n-propyl.

57. A method for hybridizing nucleic acids, comprising the steps of:

(a) providing a first nucleic acid and a second nucleic acid,

(b) incubating the nucleic acids under hybridization conditions, and

(c) identifying hybridized nucleic acids;

wherein at least one of the nucleic acids comprises a FL-nucleic-acid-Q conjugate where FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —

H, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃ where n=0 to 5, —

NO₂, —

SO₃, —

N[(CH₂)_n′

CH₃]₂ where n′

=0 to 5 or —

CN, and R₅=—

H or —

(CH₂)_n″

CH₃ where n″

=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.

58. A method in accordance with claim 57 where in the formula Q of the quencher moiety R_o is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2 position of the benzene nucleus, and R₃ and R4 are hydrogen and R₅ is ethyl.

59. A method for hybridizing nucleic acids, comprising the steps of:

(a) providing a first nucleic acid and a second nucleic acid,

(b) incubating the nucleic acids under hybridization conditions, and

(c) identifying hybridized nucleic acids;

wherein at least one of the nucleic acids comprises a FL-nucleic-acid-Q conjugate where Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and

wherein FL is a fluorophore covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, said fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —

NO₂, —

SO₃, —

C(═

O)NH₂, or —

CN;

—

OR_nn, —

SR_nn, —

OR_nn, —

NHR_nn,—

N[R_nn]₂ where R_nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;

R₁₀ and R₁₁ independently are H, —

CN, —

OR₁₂, —

N(R₁₂)₂, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃, —

NO₂, —

SO₃, —

C(═

O)NH₂, —

N[(CH₂)_nCH₃]₂ where n=0 to 5,or R₁₂ is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,;

R₁₅ is H or alkyl of 1 to 10 carbons;

R₁₆ is alkyl of 1 to 10 carbons, and

the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.

60. A method in accordance with claim 59 where the fluorophore has the formula designated FL-1.

61. A method in accordance with claim 60 where R₈ is OC(O)CH(CH₃)₂ and R₉ is H.

62. A method in accordance with claim 59 where the fluorophore has the formula designated FL-2.

63. A method in accordance with claim 62 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁ is H.

64. A method in accordance with claim 59 where the fluorophore has the formula designated FL-3.

65. A method in accordance with claim 64 where R₁₅ is methyl and R₁₆ is n-propyl.

66. A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:

(a) providing a polynucleotide comprising a target sequence,

(b) providing at least two FL-ODN-Q conjugates, wherein ODN represents an oligonucleotide moiety, one of the at least two FL-ODN-Q conjugates has a sequence that is perfectly complementary to the target sequence and at least one other of the FL-ODN-Q conjugates has a single-nucleotide mismatch with the target sequence;

(c) separately incubating each of the FL-ODN-Q conjugates with the polynucleotide under hybridization conditions; and

(d) determining the hybridization strength between each of the FL-ODN-Q and the polynucleotide, wherein FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R are independently —

H, halogen, —

O(CH₂)_nCH₃, —

(CH₂)_nCH₃ where n=0 to 5, —

NO₂, —

SO₃, —

N[(CH₂)_n′

CH₃]₂ where n′

=0 to 5 or —

CN, and R₅=—

H or —

(CH₂)_n″

CH₃ where n″

=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.

67. A method in accordance with claim 66 where in the formula of the quencher moiety Q R_o is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2 position of the benzene nucleus, and R₃ and R₄ are hydrogen and R₅ is ethyl.

68. A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:

(a) providing a polynucleotide comprising a target sequence,

(b) providing at least two FL-ODN-Q conjugates, wherein ODN represents an oligonucleotide moiety, one of the at least two FL-ODN-Q conjugates has a sequence that is perfectly complementary to the target sequence and at least one other of the FL-ODN-Q conjugates has a single-nucleotide mismatch with the target sequence;

(c) separately incubating each of the FL-ODN-Q conjugates with the polynucleotide under hybridization conditions; and

(d) determining the hybridization strength between each of the FL-ODN-Q and the polynucleotide, wherein Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and the fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,

wherein R₈and R₉ independently are H, halogen, —

NO₂, —

SO₃, —

C(═

O)NH₂, or —

CN;

—

OR_nn, —

SR_nn, —

OR_nn, —

NHR_nn,—

N[R_nn]₂ where R_nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;

R₁₀ and R₁₁ independently are H, —

CN, —

OR₁₂, —

N(R₁₂)₂, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃, —

NO₂, —

SO₃, —

C(═

O)NH₂, —

N[(CH₂)_nCH₃]₂ where n=0 to 5,or R₁₂ is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons;

R₁₅ is H or alkyl of 1 to 10 carbons;

R₁₆ is alkyl of 1 to 10 carbons, and

the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.

69. A method in accordance with claim 68 where the fluorophore has the formula designated FL-1.

70. A method in accordance with claim 69 where R₈ is OC(O)CH(CH₃)₂ and R₉ is H.

71. A method in accordance with claim 68 where the fluorophore has the formula designated FL-2.

72. A method in accordance with claim 71 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁ is H.

73. A method in accordance with claim 68 where the fluorophore has the formula designated FL-3.

74. A method in accordance with claim 73 where R₁₅ is methyl and R₁₆ is n-propyl.

75. A method for hybridizing nucleic acids, comprising the steps of:

(a) providing a first nucleic acid and a second nucleic acid,

(b) incubating the nucleic acids under hybridization conditions, and

(c) identifying hybridized nucleic acids;

wherein at least one of the nucleic acids comprises a FL-nucleic-acid-Q-MGB conjugate where FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms and Q is a quencher moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —

H, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃ where n=0 to 5, —

NO₂, —

SO₃, —

N[(CH₂)_n′

CH₃]₂ where n′

=0 to 5 or —

CN, and R₅=—

H or —

(CH₂)_n″

CH₃ where n″

=0 to 5, and where the quencher moiety is attached to the linker through the valence bond designated a.

76. A method for hybridizing nucleic acids, comprising the steps of:

(a) providing a first nucleic acid and a second nucleic acid,

(b) incubating the nucleic acids under hybridization conditions, and

(c) identifying hybridized nucleic acids;

wherein at least one of the nucleic acids comprises a FL-ODN-Q-MGB conjugate where ODN is a nucleic acid or modified nucleic acid, MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms, Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and the fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —

NO₂, —

SO₃, —

C(═

O)NH₂, or —

CN;

—

OR_nn, —

SR_n, —

OR_nn,—

NHR_nn,—

N[R_nn]₂ where R_nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;

R₁₀ and R₁₁ independently are H, —

CN, —

OR₁₂, —

N(R₁₂)₂, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃, —

NO₂, —

SO₃, —

C(═

O)NH₂, —

N[(CH₂)_nCH₃]₂ where n=0 to 5,or R₁₂ is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,

R₁₅ is H or alkyl of 1 to 10 carbons;

R₁₆ is alkyl of 1 to 10 carbons, and

the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker; and

Q comprises a diazo moiety having the formula;

wherein covalent attachment to the linker is through the nitrogen atom designated as b.

77. A method for hybridizing nucleic acids, comprising the steps of:

(a) providing a first nucleic acid and a second nucleic acid,

(b) incubating the nucleic acids under hybridization conditions, and

(c) identifying hybridized nucleic acids;

wherein at least one of the nucleic acids comprises a FL-ODN-Q-MGB conjugate where ODN is a nucleic acid or modified nucleic acid, MGB is minor groove binder moiety covalently attached to the ODN moiety or to the quencher moiety through a linker having the length of 0 to approximately 30 atoms, Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms,, and the fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —

NO₂, —

SO₃, —

C(═

O)NH₂, or —

CN;

—

OR_nn, —

SR_nn, —

OR_nn,—

NHR_nn,—

N[R_nn]₂ where R_nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;

R₁₀ and R₁₁ independently are H, —

CN, —

OR₁₂, —

N(R₁₂)₂, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃, —

NO₂, —

SO₃, —

C(═

O)NH₂, —

N[(CH₂)_nCH₃]₂ where n=0 to 5 or R₁₂ is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,

R₁₅ is H or alkyl of 1 to 10 carbons;

R₁₆ is alkyl of 1 to 10 carbons, and

the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker.

78. A method for hybridizing nucleic acids, comprising the steps of:

(a) providing a first nucleic acid and a second nucleic acid,

(b) incubating the nucleic acids under hybridization conditions, and

(c) identifying hybridized nucleic acids;

wherein at least one of the nucleic acids comprises a FL-ODN-Q conjugate where ODN is a nucleic acid or modified nucleic acid, Q is a quencher moiety covalently attached to the ODN through a linker having the length of 0 to approximately 30 atoms, and FL is a fluorophore moiety covalently attached to the nucleic acid through a linker having the length of 0 to approximately 30 atoms, and the fluorophore moiety having the structure selected from the group designated FL-1, FL-2 and FL-3 ,

wherein R₈ and R₉ independently are H, halogen, —

NO₂, —

SO₃, —

C(═

O)NH₂, or —

CN;

—

OR_nn, —

SR_nn, —

OR_nn, —

NHR_nn,—

N[R_nn2 where R_nn is independently H, an alkyl group of 1 to 10 carbons or an alkanoyl group of 1 to 10 carbons;

R₁₀ and R₁₁ independently are H, —

CN, —

OR₁₂, —

N(R₁₂)₂, halogen,—

O(CH₂)_nCH₃, —

(CH₂)_nCH₃, —

NO₂, —

SO₃, —

C(═

O)NH₂, —

N[(CH₂)_nCH₃]₂ where n=0 to 5, or R₁₂ is alkyl of 1 to 10 carbons alkanoyl of 1 to 10 carbons,

R₁₅ is H or alkyl of 1 to 10 carbons;

R₁₆ is alkyl of 1 to 10 carbons, and

the valence bond designated a symbolizes covalent attachment of the fluorophore to the linker; and

Q comprises a diazo moiety having the formula;

wherein covalent attachment to the linker is through the nitrogen atom designated as b.