Multisignal labeling reagents and processes and uses therefor

• US 20110318788A1
• Filed: 03/14/2011
• Published: 12/29/2011
• Est. Priority Date: 04/03/2003
• Status: Active Grant

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Coleman, Jack, Rabbani, Elazar, Stavrianopoulos, Jannis, Pande, Praveen
• Granted Patent: US 9,156,986 B2
• Time in Patent Office: Days
• US Class Current: 435/91.5
• CPC Class Codes: C07D 219/08 Nitrogen atoms
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07D 491/22 in which the condensed system contains four or more 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/00 Diaryl- or thriarylmethane dyes
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Provided are compounds comprising two DNA supramolecular binding molecules covalently joined by a linker group. Also provided are multisignal labeling reagents comprising (i) an oligomer of nucleotides or nucleotide analogs; (ii) a DNA supramolecular binding molecule noncovalently bound to the oligomer; and (iii) a first reactive group or a first partner of a first binding pair covalently bound to the oligomer. Additionally provided are methods of producing multisignal labeling reagents.

Claims

1. A compound comprising two DNA supramolecular binding molecules covalently joined by a linker group, the compound further comprising a detectable label bound thereto, wherein the detectable label is not either of the two DNA supramolecular binding molecules.

2. The compound of claim 1, wherein at least one of the DNA supramolecular binding molecules is a minor groove binder.

3. The compound of claim 2, wherein the minor groove binder is a diacrylamidine, or a bis-benzimidazole.

4. The compound of claim 2, wherein the minor groove binder is DAPI, berenil, pentamidine, distamycin A, or Hoechst 33258.

5. The compound of claim 1, wherein at least one of the DNA supramolecular binding molecules is a major groove binder.

6. The compound of claim 5, wherein the major groove binder comprises a zinc finger, a leucine zipper or a helix-turn-helix motif.

7. The compound of claim 1, wherein at least one of the DNA supramolecular binding molecules is an intercalator.

8. The compound of claim 1, wherein both of the DNA supramolecular binding molecules are intercalators.

9. The compound of claim 8, wherein the two DNA intercalators are the same.

10. The compound of claim 8, wherein the two DNA intercalators are different intercalators.

11. The compound of claim 7, wherein at least one of the two DNA intercalators is an acridine, a coumarin, a psoralen, a phenanthridine, a quinoxaline, an anthracycline, or a metallo-intercalator.

12. The compound of claim 7, wherein at least one of the two DNA intercalators is 9-aminoacridine, 7-hydroxy-4-methylcoumarin, 7-amino-4-methylcoumarin, 4-methyl-7-sulphato-methylcoumarin, 8-[[[(diethylamino)methyl]propyl]oxy]psoralen, 5(N-piperadinyl)-8-methoxypsoralen, ethidium bromide, thiazole orange, 6-(4′

-carboxyphenyl)-3,8-diamine-5-methyl phenanthridinium chloride, doxorubicin, daunomycin, [Pt(tpy)(SCH₂CH₂OH)]⁺, or [Rh(phi)(Me₂-trien)]³⁺.

13. The compound of claim 1, wherein the linker is flexible.

14. The compound of claim 1, wherein the linker is an unsubstituted C₁-C₂₀ straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group, a substituted C₁-C₂₀ straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group wherein one or more C, CH or CH₂ groups are substituted with an O atom, N atom, S atom, NH group, CO group or OCO group, or an unsubstituted or substituted aromatic group.

15. The compound of claim 1, wherein the linker is —

(CH₂)_1-10—

NH—

(CH₂)_1-10—

.

16. The compound of claim 1, wherein the linker is —

(CH₂)_1-5—

NH—

(CH₂)_1-5—

.

17. The compound of claim 1, wherein the linker is —

(CH₂)₃—

NH—

(CH₂)₄—

.

18. The compound of claim 1, wherein the detectable label is radioactive.

19. The compound of claim 1, wherein the detectable label is non-radioactive.

20. The compound of claim 19, wherein the detectable label is a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, or an energy transfer compound.

21. The compound of claim 19, wherein the non-radioactive detectable label is a fluorophore.

22. The compound of claim 21, wherein the fluorophore comprises a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin or an iminocoumarin.

23. The compound of claim 21, wherein the fluorophore comprises a xanthene.

24. The compound of claim 23, wherein the xanthene is a rhodamine or rhodamine derivative.

25. The compound of claim 23, wherein the xanthene is a rosamine or rosamine derivative.

26. The compound of claim 23, wherein the xanthene is a rhodol or rhodol derivative.

27. The compound of claim 21, wherein the fluorophore comprises a coumarin.

28. The compound of claim 27, wherein the coumarin is an iminocoumarin.

29. The compound of claim 19, wherein the non-radioactive detectable label is a chemiluminescent or phosphorescent moiety.

30. The compound of claim 1, wherein the detectable label is non-covalently bound to the compound via a binding pair.

31. The compound of claim 1, wherein the detectable label is covalently bound to the compound.

32. The compound of claim 31, wherein the detectable label is bound to the linker group.

33. The compound of claim 1, further comprising a third intercalator covalently joined by a linker group.

34. The compound of claim 1, wherein the compound comprises

56. The multisignal labeling reagent of claim 35, comprising the compound of any one of claims 1-34.

35. A multisignal labeling reagent comprising

(i) an oligomer of nucleotides or nucleotide analogs;

(ii) a DNA supramolecular binding molecule noncovalently bound to the oligomer; and

(iii) a covalently bound first reactive group or a first partner of a first binding pair.

36. The multisignal labeling reagent of claim 35, wherein the DNA supramolecular binding molecule is fluorescent.

37. The multisignal labeling reagent of claim 35, wherein the DNA supramolecular binding molecule further comprises a detectable label that is not the supramolecular binding molecule.

38. The multisignal labeling reagent of claim 37, wherein the detectable label is non-radioactive.

39. The multisignal labeling reagent of claim 38, wherein the non-radioactive detectable label is a fluorophore.

40. The multisignal labeling reagent of claim 38, wherein the non-radioactive detectable label is a chemiluminescent or phosphorescent moiety.

41. The multisignal labeling reagent of claim 35, wherein the DNA supramolecular binding molecule is a minor groove binder.

42. The multisignal labeling reagent of claim 41, wherein the minor groove binder is a diacrylamidine, or a bis-benzimidazole.

43. The multisignal labeling reagent of claim 41, wherein the minor groove binder is DAPI, berenil, pentamidine, distamycin A, or Hoechst 33258.

44. The multisignal labeling reagent of claim 35, wherein the DNA supramolecular binding molecule is a major groove binder.

45. The multisignal labeling reagent of claim 44, wherein the major groove binder comprises a zinc finger, a leucine zipper or a helix-turn-helix motif.

46. The multisignal labeling reagent of claim 35, wherein the DNA supramolecular binding molecule is an intercalator.

47. The multisignal labeling reagent of claim 46, wherein the intercalator is acridine, a coumarin, a psoralen, a phenanthridine, an anthracycline, or a metallo-intercalator.

48. The multisignal labeling reagent of claim 46, wherein the intercalator is 9-aminoacridine, 7-hydroxy-4-methylcoumarin, 7-amino-4-methylcoumarin, 4-methyl-7-sulphato-methylcoumarin, 8-[[[(diethylamino)methyl]propyl]oxy]psoralen, 5(N-piperadinyl)-8-methoxypsoralen, ethidium bromide, 6-(-4′

-carboxyphenyl)-3,8-diamine-5-methyl phenanthridinium chloride, doxorubicin, daunomycin, [Pt(tpy)(SCH₂CH₂OH)]⁺, or [Rh(phi)(Me₂-trien)]³⁺.

49. The multisignal labeling reagent of claim 35, wherein the multisignal labeling reagent comprises a first partner of a first binding pair.

50. The multisignal labeling reagent of claim 49, wherein the first binding pair is a ligand/receptor, a hormone/receptor, biotin/avidin, biotin/streptavidin or an antigen/antibody.

51. The multisignal labeling reagent of claim 49, wherein the first partner of the first binding pair is streptavidin.

52. The multisignal labeling reagent of claim 35, wherein the primer further comprises the first reactive group or the first binding partner of the first binding pair.

53. The multisignal labeling reagent of claim 35, wherein the multisignal labeling reagent further comprises a polymer to which two or more of the oligomers are bound, wherein the first reactive group or the first partner of the first binding pair is covalently attached to the polymer.

54. The multisignal labeling reagent of claim 53, wherein the two or more oligomers are bound to the polymer covalently.

55. The multisignal labeling reagent of claim 53, wherein

the polymer is a nucleic acid and

the two or more oligomers are bound to the polymer by complementary hybridization.

57. A method of producing a multisignal labeling reagent, the method comprising

(a) obtaining

(i) a primer comprising an oligonucleotide and a first reactive group or a first partner of a first binding pair at the 5′

end of the oligonucleotide;

(ii) a template comprising a nucleic acid comprising a first sequence that is complementary to the oligonucleotide and a second sequence that extends in the 5′

direction from the first sequence;

(iii) a polymerase capable of extending the oligonucleotide along the template nucleic acid when the template nucleic acid is hybridized to the oligonucleotide at the first sequence; and

(iv) nucleotide triphosphates (NTPs) or analogs thereof that are capable of being incorporated into the extended oligonucleotide, wherein at least one of the NTPs or analogs comprises a non-radioactive detectable label, a second reactive group or a first partner of a second binding pair; and

(b) combining the primer, template, polymerase and NTPs or analogs under conditions such that the oligonucleotide hybridizes to the first sequence and is extended along the second sequence, where the extended oligonucleotide comprises at least two NTPs or analogs incorporated therein that comprise a non-radioactive detectable label, a second reactive group or a first partner of a second binding pair;

wherein,

(A) if at least one of the two or more NTPs or analogs incorporated into the extended oligonucleotide comprises a second reactive group, the method further comprises combining the extended oligonucleotide with a first compound comprising a non-radioactive detectable label covalently linked to a moiety capable of reacting with the second reactive group such that the label is covalently linked to the extended primer, and

(B) if at least one of the two or more NTPs or analogs incorporated into the extended oligonucleotide comprises a first partner of the second binding pair, the method further comprises combining the extended primer with a second compound comprising the non-radioactive detectable label covalently linked to a second binding partner of the second binding pair.

58. The method of claim 57, wherein the non-radioactive detectable labels are fluorophores, phosphorescent moieties, chemiluminescent moieties, chelating moieties, electron dense moieties, magnetic moieties, or energy transfer moieties.

59. The method of claim 57, wherein the non-radioactive detectable labels are fluorophores.

60. The method of claim 59, wherein the fluorophore comprises a symmetric or asymmetric cyanine, a merocyanine, a styryl moiety, an oxazine, a xanthene, a coumarin or an iminocoumarin.

61. The method of claim 57, wherein the fluorophore comprises a xanthene.

62. The method of claim 61, wherein the xanthene is a rhodamine or rhodamine derivative.

63. The method of claim 61, wherein the xanthene is a rosamine or rosamine derivative.

64. The method of claim 61, wherein the xanthene is a rhodol or rhodol derivative.

65. The method of claim 59, wherein the fluorophore comprises a coumarin.

66. The method of claim 59, wherein the coumarin is an iminocoumarin.

67. The method of claim 57, wherein the non-radioactive detectable labels are chemiluminescent or phosphorescent moieties.

68. The method of claim 67, wherein the NTP or NTP analogs are each labeled with a non-radioactive detectable label.

69. The method of claim 57, wherein the two or more NTPs or analogs are each labeled with a reactive group.

70. The method of claim 69, wherein each reactive group is joined to the NTPs by a linker arm.

71. The method of claim 69, wherein the reactive group is an isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal, aldehyde, mercury salt, or any combination thereof.

72. The method of claim 57, wherein the NTP or NTP analogs are each labeled with a first binding partner of a second binding pair.

73. The method of claim 72, wherein the first binding partner of the second binding pair is a biotin.

74. The method of claim 71, wherein each first binding partner of the second binding pair is connected to the NTPs by a linker arm.

75. The method of claim 57, wherein the multisignal labeling reagent further comprises a non-inherent charged group that increases the aqueous solubility of the reagent.

76. The method of claim 75, wherein the charged group is a phosphate, a carboxylic acid, a sulfone, an amine or a hydroxy group.

77. The method of claim 57, wherein the primer comprises a first partner of a first binding pair.

78. The method of claim 77, wherein the first binding pair is a ligand/receptor, a hormone/receptor, biotin/avidin, biotin/streptavidin or an antigen/antibody.

79. The method of claim 77, wherein the first partner of the first binding pair is streptavidin.

80. The method of claim 57, further comprising combining the multisignal labeling reagent with a target molecule such that the multisignal labeling reagent is bound to the target by the first reactive group or the first partner of the first binding pair.

81. A method of producing a multisignal labeling reagent, the method comprising

(a) obtaining

(i) a primer comprising an oligonucleotide;

(ii) a template comprising a nucleic acid comprising a first sequence that is complementary to the oligonucleotide and a second sequence that extends in the 5′

direction from the first sequence;

(iii) a polymerase capable of extending the oligonucleotide along the template nucleic acid when the template nucleic acid is hybridized to the oligonucleotide at the first sequence;

(iv) nucleotide triphosphates (NTPs) or analogs thereof that are capable of being incorporated into the extended oligonucleotide, wherein at least one of the NTPs or analogs comprises a non-radioactive detectable label, a second reactive group or a first partner of a second binding pair; and

(v) a polymer capable of binding to more than one of the extended oligonucleotide, wherein the polymer comprises a first reactive group or a first partner of a first binding pair;

(b) combining the primer, template, polymerase and NTPs or analogs under conditions such that the oligonucleotide hybridizes to the first sequence and is extended along the second sequence, where the extended oligonucleotide comprises at least two NTPs or analogs incorporated therein that comprise a non-radioactive detectable label, a second reactive group or a first partner of a second binding pair; and

(c) combining the extended oligonucleotide with the polymer under conditions such that at least two of the extended oligonucleotides bind to the polymer,

wherein,

(A) if at least one of the two or more NTPs or analogs incorporated into the extended oligonucleotide comprises a second reactive group, the method further comprises combining the extended oligonucleotide with a first compound comprising a non-radioactive detectable label covalently linked to a moiety capable of reacting with the second reactive group such that the label is covalently linked to the extended primer, and

(B) if at least one of the two or more NTPs or analogs incorporated into the extended oligonucleotide comprises a first partner of the second binding pair, the method further comprises combining the extended primer with a second compound comprising the non-radioactive detectable label covalently linked to a second binding partner of the second binding pair.

82. The method of claim 81, wherein the non-radioactive detectable labels are fluorophores, phosphorescent moieties, chemiluminescent moieties, chelating moieties, electron dense moieties, magnetic moieties, or energy transfer moieties.

83. The method of claim 81, wherein the non-radioactive detectable labels are fluorophores.

84. The method of claim 81, wherein the two or more NTPs or analogs are each labeled with a reactive group.

85. The method of claim 84, wherein each reactive group is joined to the NTPs by a linker arm.

86. The method of claim 81, wherein the NTP or NTP analogs are each labeled with a non-radioactive detectable label.

87. The method of claim 83, wherein the NTP or NTP analogs are each labeled with a first binding partner of a second binding pair.

88. The method of claim 87, wherein the first binding partner of the second binding pair is a biotin.

89. The method of claim 87, wherein each first binding partner of the second binding pair is connected to the NTPs by a linker arm.

90. The method of claim 81, wherein the multisignal labeling reagent further comprises a non-inherent charged group that increases the aqueous solubility of the reagent.

91. The method of claim 81, wherein the polymer comprises a first partner of a first binding pair.

92. The method of claim 91, wherein the first binding pair is a ligand/receptor, a hormone/receptor, biotin/avidin, biotin/streptavidin or an antigen/antibody.

93. The method of claim 91, wherein the first partner of the first binding pair is biotin.

94. The method of claim 82, wherein the polymer is a polynucleotide comprising more than one region that is complementary to a portion of the extended oligonucleotide

95. The method of claim 81, further comprising combining the multisignal labeling reagent with a target molecule such that the multisignal labeling reagent is bound to the target by the first reactive group or the first partner of the first binding pair.