Oligo- or polynucleotides comprising phosphate-moiety labeled nucleotides

• US 6,992,180 B1
• Filed: 06/07/1995
• Issued: 01/31/2006
• Est. Priority Date: 06/23/1982
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Rabbani, Elazar, Kirtikar, Dollie, Kline, Stanley, Engelhardt, Dean, Stavrianopoulos, Janes G.
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US08/479,997
• Time in Patent Office: 3,891 Days
• Field of Search: 435/6, 435/7.1, 536/26.6, 536/25.31, 536/25.32, 536/23.1
• US Class Current: 536/23.1
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: C07B 59/008 Peptides; Proteins
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07K 17/06 attached to the carrier via a bridging agent
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/117 incorporating modified base

Abstract

The present invention provides a nucleotide having the formula,
Sig-PM-SM-BASE
wherein PM is a phosphate moiety, SM is a sugar moiety and BASE is a pyrimidine, purine or 7-deazapurine moiety. PM is attached to the 3′ or the 5′ position of the sugar moiety when the nucleotide is a deoxyribonucleotide and at the 2′, 3′ or 5′ position when the nucleotide is a ribonucleotide. BASE is attached to the 1′ position of SM from the N¹ position when BASE is a pyrmidine or the N⁹ position when BASE is a purine or 7-deazapurine. Sig is covalently attached to PM directly or via a chemical linkage, and represents a detectable moiety covalently attached to SM directly or through a linkage group. Also provided are an oligo- or polynucleotide comprising at least one such phosphate-moiety labeled nucleotide, and other compositions including those wherein a polypeptide is terminally ligated or attached to the oligo- or polynucleotide. The phosphate-moiety labeled nucleotide, and the oligo- or polynucleotides and other compositions comprising such phosphate-moiety labeled nucleotides, are useful as diagnostic tools for detecting analytes and as therapeutic agents.

Claims

1. An oligo- or polynucleotide which is complementary to a nucleic acid of interest or a portion thereof, said oligo- or polynucleotide comprising at least one modified nucleotide or modified nucleotide analog having the formula

Sig-PM-SM-BASE

wherein PM is a phosphate moiety, SM is a furanosyl moiety and BASE is a base moiety comprising a pyrimidine, a pyrimidine analog, a purine, a purine analog, a deazapurine or a deazapurine analog wherein said analog can be attached to or coupled to or incorporated into DNA or RNA wherein said analog does not substantially interfere with double helix formation or nucleic acid hybridization, said PM being attached to SM, said BASE being attached to SM, and said Sig being covalently attached to PM directly or through a non-nucleotidyl chemical linkage, and wherein said Sig comprises a non-polypeptide, non-nucleotidyl, non-radioactive label moiety which can be directly or indirectly detected when attached to PM or when said modified nucleotide is incorporated into said oligo- or polynucleotide or when said oligo- or polynucleotide is hybridized to said complementary nucleic acid of interest or a portion thereof, and wherein Sig comprises biotin, iminobiotin, an electron dense component, a magnetic component, a metal-containing component, a fluorescent component, a chemiluminescent component, a chromogenic component, a hapten or a combination of any of the foregoing.

2. The oligo- or polynucleotide of claim 1, wherein Sig comprises at least three carbon atoms.

3. The oligo- or polynucleotide of claim 1, wherein said magnetic component comprises magnetic oxide.

4. The oligo- or polynucleotide of claim 3, wherein said magnetic oxide comprises ferric oxide.

5. The oligo- or polynucleotide of claim 1, wherein said metal-containing component is catalytic.

6. The oligo- or polynucleotide of claim 1, wherein said fluorescent component comprises fluorescein, rhodamine or dansyl.

7. The oligo- or polynucleotide of claim 1, wherein said covalent attachment comprises

8. The oligo- or polynucleotide of claim 1, wherein said chemical linkage does not interfere substantially with the characteristic ability of Sig to form a detectable signal.

9. The oligo- or polynucleotide of claim 1, wherein said chemical linkage comprises a —

CH₂NH—

moiety.

10. The oligo- or polynucleotide of claim 1, wherein said chemical linkage comprises an allylamine group.

11. The oligo- or polynucleotide of claim 1, wherein said chemical linkage comprises any of the moieties:

12. The oligo- or polynucleotide of claim 1, wherein said PM comprises a monophosphate, a diphosphate or a triphosphate.

13. The oligo- or polynucleotide of claim 1, wherein said Sig moiety is covalently attached to said PM through a phosphorous atom or phosphate oxygen.

14. The oligo- or polynucleotide of claim 1, wherein said Sig moiety is attached to the PM of a terminal nucleotide in said oligo- or polydeoxyribonucleotide.

15. The oligo- or polynucleotide of claim 14, wherein the furanosyl moiety of said terminal nucleotide comprises a hydrogen atom at the 2′

position thereof.

16. The oligo- or polynucleotide of claim 14, wherein the furanosyl moiety of said terminal nucleotide comprises an oxygen atom at the 2′

position thereof.

17. The oligo- or polynucleotide of claim 15, wherein the furanosyl moiety of said terminal nucleotide comprises a hydrogen atom at the 3′

position thereof.

18. The oligo- or polynucleotide of claim 16, wherein the furanosyl moiety of said terminal nucleotide comprises an oxygen atom at the 3′

position thereof.

19. The oligo- or polynucleotide of claim 1, wherein said furanosyl moiety comprises a ribose, a deoxyribose or a dideoxyribose.

20. The oligo- or polynucleotide of claim 1, wherein said pyrimidine analogs comprise thymidine analogs, uridine analogs, deoxyuridine analogs, cytidine analogs, deoxycytidine analogs or a combination of any of the foregoing.

21. The oligo- or polynucleotide of claim 20, wherein said uridine analogs comprise 5-bromo-2′

-deoxyuridine-5′

-phosphate.

22. The oligo- or polynucleotide of claim 20, wherein said deoxycytidine analogs comprise 5-hydroxymethyl-2′

-deoxycytidylic acid.

23. The oligo- or polynucleotide of claim 1, wherein said purine analogs comprise adenosine analogs, deoxyadenosine analogs, guanosine analogs, deoxyguanosine analogs or a combination of any of the foregoing.

24. The oligo- or polynucleotide of claim 23, wherein said adenosine analogs comprise tubericidin and toyocamycin.

25. The oligo- or polynucleotide of claim 1, wherein said oligo- or polynucleotide comprises an oligo- or polydeoxyribonucleotide.

26. The oligo- or polynucleotide of claim 1, wherein said oligo- or polynucleotide comprises an oligo- or polydeoxyribonucleotide and further comprises at least one ribonucleotide.

27. The oligo- or polynucleotide of claim 1, wherein said oligo- or polynucleotide comprises an oligo- or polyribonucleotide.

28. The oligo- or polynucleotide of claim 1, wherein said oligo- or polynucleotide comprises an oligo- or polyribonucleotide and further comprises at least one deoxyribonucleotide.

29. An oligo- or polynucleotide which is complementary to a nucleic acid of interest or a portion thereof, said oligo- or polynucleotide comprising at least one modified nucleotide or a modified nucleotide analog having the structural formula:

wherein BASE is a moiety comprising a pyrimidine, a pyrimidine analog, a purine, a purine analog, a deazapurine or a deazapurine analog, wherein said analog can be attached to or coupled to or incorporated into DNA or RNA, wherein said analog does not substantially interfere with double helix formation or nucleic acid hybridization, and wherein said BASE is attached to the 1′

position of the furanosyl ring from the N1 position when said BASE is a pyrimidine or a pyrimidine analog, or from the N9 position when said BASE is a purine, a purine analog, a deazapurine or a deazapurine analog;

wherein x comprises H—

, HO—

, a mono-phosphate, a di-phosphate or a tri-phosphate;

wherein y comprises H—

, HO—

, a mono-phosphate, a di-phosphate or a tri-phosphate;

wherein z comprises H—

, HO—

, a mono-phosphate, a di-phosphate or a tri-phosphate; and

wherein Sig is covalently attached directly or through a non-nucleotidyl chemical linkage to at least one phosphate comprising x, y, z, or a combination thereof, and wherein said Sig comprises a non-polypeptide, non-nucleotidyl, non-radioactive label moiety which can be directly or indirectly detected when attached to said phosphate or when said modified nucleotide is incorporated into said oligo- or polynucleotide or when said oligo- or polynucleotide is hybridized to said complementary nucleic acid of interest or a portion thereof, wherein Sig comprises biotin, iminobiotin, an electron dense component, a magnetic component, a metal-containing component, a fluorescent component, a chemiluminescent component, a chromogenic component, a hapten, or a combination of any of the foregoing.

30. The oligo- or polynucleotide of claim 29, wherein Sig comprises at least three carbon atoms.

31. The oligo- or polynucleotide of claim 29, wherein said magnetic component comprises magnetic oxide.

32. The oligo- or polynucleotide of claim 31, wherein said magnetic oxide comprises ferric oxide.

33. The oligo- or polynucleotide of claim 29, wherein said metal-containing component is catalytic.

34. The oligo- or polynucleotide of claim 29, wherein said fluorescent component comprises fluorescein, rhodamine or dansyl.

35. The oligo- or polynucleotide of claim 29, wherein said covalent attachment comprises

36. The oligo- or polynucleotide of claim 29, wherein said chemical linkage does not interfere substantially with the characteristic ability of Sig to form a detectable signal.

37. The oligo- or polynucleotide of claim 29, wherein said chemical linkage comprises a —

CH₂NH—

moiety.

38. The oligo- or polynucleotide of claim 29, wherein said chemical linkage comprises an allylamine group.

39. The oligo- or polynucleotide of claim 29, wherein said chemical linkage comprises any of the moieties:

40. The oligo- or polynucleotide of claim 29, wherein said x comprises a monophosphate, a diphosphate or a triphosphate and y comprises a monophosphate.

41. The oligo- or polynucleotide of claim 29, wherein said Sig moiety is covalently attached to said phosphate through a phosphorus atom or phosphate oxygen.

42. The oligo- or polynucleotide of claim 29, wherein said x comprises a monophosphate.

43. The oligo- or polynucleotide of claim 29, wherein said Sig moiety is attached to the phosphate moiety of a terminal nucleotide in said oligo- or polynucleotide.

44. The oligo- or polynucleotide of claim 43, wherein z of said furanosyl moiety of said terminal nucleotide comprises a hydrogen atom.

45. The oligo- or polynucleotide of claim 43, wherein z of said furanosyl moiety of said terminal nucleotide comprises an oxygen atom.

46. The oligo- or polynucleotide of claim 44, wherein y of said furanosyl moiety comprises a hydrogen atom.

47. The oligo- or polynucleotide of claim 45, wherein y of said furanosyl moiety comprises an oxygen atom.

48. The oligo- or polynucleotide of claim 29, wherein said furanosyl moiety comprises a ribose, a deoxyribose or a dideoxyribose.

49. The oligo- or polynucleotide of claim 29, wherein said pyrimidine analogs comprise thymidine analogs, uridine analogs, deoxyuridine analogs, cytidine analogs, deoxycytidine analogs or a combination of any of the foregoing.

50. The oligo- or polynucleotide of claim 49, wherein said uridine analogs comprise 5-bromo-2′

-deoxyuridine-5′

-phosphate.

51. The oligo- or polynucleotide of claim 49, wherein said deoxycytidine analogs comprise 5-hydroxymethyl-2′

-deoxycytidylic acid.

52. The oligo- or polynucleotide of claim 29, wherein said purine analogs comprise adenosine analogs, deoxyadenosine analogs, guanosine analogs, deoxyguanosine analogs, or a combination of any of the foregoing.

53. The oligo- or polynucleotide of claim 52, wherein said adenosine analogs comprise tubericidin or toyocamycin.

54. The oligo- or polynucleotide of claim 29, wherein said oligo- or polynucleotide comprises an oligo- or polydeoxyribonucleotide.

55. The oligo- or polynucleotide of claim 29, wherein said oligo- or polynucleotide comprises an oligo- or polydeoxyribonucleotide and further comprises at least one ribonucleotide.

56. The oligo- or polynucleotide of claim 29, wherein said oligo- or polynucleotide comprises an oligo- or polyribonucleotide.

57. The oligo- or polynucleotide of claim 29, wherein said oligo- or polynucleotide comprises an oligo- or polyribonucleotide and further comprises at least one deoxyribonucleotide.

58. The oligo- or polynucleotide of claim 29, having the structural formula:

wherein m and n represent integers from 0 up to about 100,000, and wherein said Sig moiety is attached to at least one of the phosphate moieties in said structural formula.

59. An oligo- or polynucleotide which is complementary to a nucleic acid of interest or a portion thereof, said oligo- or polynucleotide comprising at least one modified nucleotide or a modified nucleotide analog having the formula

Sig-PM-SM-BASE

wherein PM is a phosphate moiety, SM is a furanosyl moiety and BASE is a base moiety comprising a pyrimidine, a pyrimidine analog, a purine, a purine analog, a deazapurine or a deazapurine analog, wherein said analog can be attached to or coupled to or incorporated into DNA or RNA, wherein said analog does not substantially interfere with double helix formation or nucleic acid hybridization, said PM is attached to SM, said BASE is attached to SM, said Sig is covalently attached to PM directly or via a non-nucleotidyl chemical linkage, and wherein said Sig comprises a non-polypeptide, non-nucleotidyl, non-radioactive label moiety which can be directly or indirectly detected when attached to PM or when said modified nucleotide is incorporated into said oligo- or polynucleotide, or when said oligo- or polynucleotide is hybridized to said complementary nucleic acid of interest or a portion thereof, provided that when said oligo- or polynucleotide is an oligoribonucleotide or a polyribonucleotide, and when Sig is attached through a chemical linkage to a terminal PM at the 3′

position of a terminal ribonucleotide, said chemical linkage is not obtained through a 2′

,3′

vicinal oxidation of a 3′

terminal ribonucleotide previously attached to said oligoribonucleotide or polyribonucleotide, and wherein said Sig comprises biotin, iminobiotin, an electron dense component, a magnetic component, a metal-containing component, a fluorescent component, a chemiluminescent component, a chromogenic component, a hapten, or a combination of any of the foregoing.

60. The oligo- or polynucleotide of claim 59, wherein Sig comprises at least three carbon atoms.

61. The oligo- or polynucleotide of claim 59, wherein said magnetic component comprises magnetic oxide.

62. The oligo- or polynucleotide of claim 61, wherein said magnetic oxide comprises ferric oxide.

63. The oligo- or polynucleotide of claim 59, wherein said metal-containing component is catalytic.

64. The oligo- or polynucleotide of claim 59, wherein said fluorescent component comprises fluorescein, rhodamine or dansyl.

65. The oligo- or polynucleotide of claim 59, wherein said covalent attachment comprises

66. The oligo- or polynucleotide of claim 59, wherein said chemical linkage does not interfere substantially with the characteristic ability of Sig to form a detectable signal.

67. The oligo- or polynucleotide of claim 59, wherein said chemical linkage comprises a —

CH₂NH—

moiety.

68. The oligo- or polynucleotide of claim 59, wherein said chemical linkage comprises an allylamine group.

69. The oligo- or polynucleotide of claim 59, wherein said chemical linkage comprises any of the moieties:

70. The oligo- or polynucleotide of claim 59, wherein said PM comprises a monophosphate, a diphosphate or a triphosphate.

71. The oligo- or polynucleotide of claim 59, wherein said Sig moiety is covalently attached to said PM through a phosphorus atom or phosphate oxygen.

72. The oligo- or polynucleotide of claim 59, wherein said Sig moiety is attached to the PM of a terminal nucleotide in said oligo- or polynucleotide.

73. The oligo- or polynucleotide of claim 72, wherein the furanosyl moiety of said terminal nucleotide has a hydrogen atom at the 2′

position thereof.

74. The oligo- or polynucleotide of claim 72, wherein the furanosyl moiety of said terminal nucleotide has an oxygen atom at the 2′

position thereof.

75. The oligo- or polynucleotide of claim 73, wherein the furanosyl moiety of said terminal nucleotide has a hydrogen atom at the 3′

position thereof.

76. The oligo- or polynucleotide of claim 74, wherein the furanosyl moiety of said terminal nucleotide has an oxygen atom at the 3′

position thereof.

77. The oligo- or polynucleotide of claim 59, wherein said furanosyl moiety comprises a ribose, a deoxyribose or a dideoxyribose.

78. The oligo- or polynucleotide of claim 59, wherein said pyrimidine analogs comprise thymidine analogs, uridine analogs, deoxyuridine analogs, cytidine analogs, deoxycytidine analogs or a combination of any of the foregoing.

79. The oligo- or polynucleotide of claim 78, wherein said uridine analogs comprise 5-bromo-2′

-deoxyuridine-5′

-phosphate.

80. The oligo- or polynucleotide of claim 78, wherein said deoxycytidine analogs comprise 5-hydroxymethyl-2′

-deoxycytidylic acid.

81. The oligo- or polynucleotide of claim 59, wherein said purine analogs comprise adenosine analogs, deoxyadenosine analogs, guanosine analogs, deoxyguanosine analogs or a combination of any of the foregoing.

82. The oligo- or polynucleotide of claim 81, wherein said adenosine analogs comprise tubericidin and toyocamycin.

83. The oligo- or polynucleotide of claim 59, wherein said oligo- or polynucleotide comprises an oligo- or polydeoxyribonucleotide.

84. The oligo- or polynucleotide of claim 59, wherein said oligo- or polynucleotide comprises an oligo- or polydeoxyribonucleotide and further comprises at least one ribonucleotide.

85. The oligo- or polynucleotide of claim 59, wherein said oligo- or polynucleotide comprises an oligo- or polyribonucleotide.

86. The oligo- or polynucleotide of claim 59, wherein said oligo- or polynucleotide comprises an oligo- or polyribonucleotide and further comprises at least one deoxyribonucleotide.

87. An oligo- or polynucleotide which is complementary to a nucleic acid of interest or a portion thereof, said oligo- or polynucleotide comprising at least one modified nucleotide or a modified nucleotide analog having the structural formula:

wherein BASE is a moiety comprising a pyrimidine, a pyrimidine analog, a purine, a purine analog, a deazapurine, a deazapurine analog, wherein said analog can be attached to or coupled to or incorporated into DNA or RNA wherein said analog does not substantially interfere with double helix formation or nucleic acid hybridization, and wherein BASE is attached to the 1′

position of the furanosyl ring from the N1 position when BASE is a pyrimidine or a pyrimidine analog, from the N9 position of the furanosyl ring when BASE is a purine, a purine analog, a deazapurine or a deazapurine;

wherein x comprises H—

, HO—

, a mono-phosphate, a di-phosphate or a tri-phosphate;

wherein y comprises H—

, HO—

, a mono-phosphate, a di-phosphate or a tri-phosphate;

wherein z comprises H—

, HO—

, a mono-phosphate, a di-phosphate or a tri-phosphate; and

wherein said Sig is covalently attached directly or through a non-nucleotidyl chemical linkage to at least one phosphate comprising of x, y and z, or a combination thereof, and wherein said Sig comprises a non-polypeptide, non-nucleotidyl, non-radioactive label moiety which can be directly or indirectly detected when so attached to said phosphate or when said modified nucleotide is incorporated into said oligo- or polynucleotide, or when said oligo- or polynucleotide is hybridized to said complementary nucleic acid of interest or a portion thereof, provided that when said oligo- or polynucleotide is an oligoribonucleotide or a polyribonucleotide and when Sig is attached through a chemical linkage to a terminal PM at the 3′

position of a terminal ribonucleotide, said chemical linkage is not obtained through a 2′

,3′

vicinal oxidation of a 3′

terminal ribonucleotide previously attached to said oligoribonucleotide or polyribonucleotide, and wherein Sig comprises biotin, iminobiotin, an electron dense component, a magnetic component, a metal-containing component, a fluorescent component, a chemiluminescent component, a chromogenic component, a hapten, or a combination of any of the foregoing.

88. The oligo- or polynucleotide of claim 87, wherein Sig comprises at least three carbon atoms.

89. The oligo- or polynucleotide of claim 87, wherein said magnetic component comprises magnetic oxide.

90. The oligo- or polynucleotide of claim 89, wherein said magnetic oxide comprises ferric oxide.

91. The oligo- or polynucleotide of claim 87, wherein said metal-containing component is catalytic.

92. The oligo- or polynucleotide of claim 87, wherein said fluorescent component comprises fluorescein, rhodamine or dansyl.

93. The oligo- or polynucleotide of claim 87, wherein said covalent attachment comprises

94. The oligo- or polynucleotide of claim 87, wherein said chemical linkage does not interfere substantially with the characteristic ability of Sig to form a detectable signal.

95. The oligo- or polynucleotide of claim 87, wherein said chemical linkage comprises a —

CH₂NH—

moiety.

96. The oligo- or polynucleotide of claim 87, wherein said chemical linkage comprises an allylamine group.

97. The oligo- or polynucleotide of claim 87, wherein said chemical linkage comprises any of the moieties:

98. The oligo- or polynucleotide of claim 87, wherein said x comprises a monophosphate, a diphosphate or a triphosphate and y comprises a monophosphate.

99. The oligo- or polynucleotide of claim 87, wherein said Sig moiety is covalently attached to said phosphate through a phosphorus atom or phosphate oxygen.

100. The oligo- or polynucleotide of claim 87, wherein said x comprises a monophosphate.

101. The oligo- or polynucleotide of claim 87, wherein said Sig moiety is attached to the phosphate moiety of a terminal nucleotide in said oligo- or polynucleotide.

102. The oligo- or polynucleotide of claim 101, wherein z of said furanosyl moiety of said terminal nucleotide comprises a hydrogen atom.

103. The oligo- or polynucleotide of claim 101, wherein z of said furanosyl moiety of said terminal nucleotide comprises an oxygen atom.

104. The oligo- or polynucleotide of claim 102, wherein y of said furanosyl moiety comprises a hydrogen atom.

105. The oligo- or polynucleotide of claim 103, wherein y of said furanosyl moiety comprises an oxygen atom.

106. The oligo- or polynucleotide of claim 87, wherein said furanosyl moiety comprises a ribose, a deoxyribose or a dideoxyribose.

107. The oligo- or polynucleotide of claim 87, wherein said pyrimidine analogs comprise thymidine analogs, uridine analogs, deoxyuridine analogs, cytidine analogs, deoxycytidine analogs or a combination of any of the foregoing.

108. The oligo- or polynucleotide of claim 107, wherein said uridine analogs comprise 5-bromo-2′

-deoxyuridine-5′

-phosphate.

109. The oligo- or polynucleotide of claim 107, wherein said deoxycytidine analogs comprise 5-hydroxymethyl-2′

-deoxycytidylic acid.

110. The oligo- or polynucleotide of claim 87, wherein said purine analogs comprise adenosine analogs, deoxyadenosine analogs, guanosine analogs, deoxyguanosine analogs, or a combination of any of the foregoing.

111. The oligo- or polynucleotide of claim 110, wherein said adenosine analogs comprise tubericidin or toyocamycin.

112. The oligo- or polynucleotide of claim 87, wherein said oligo- or polynucleotide comprises an oligo- or polydeoxyribonucleotide.

113. The oligo- or polynucleotide of claim 112, wherein said oligo- or polynucleotide comprises an oligo- or polydeoxyribonucleotide and further comprises at least one ribonucleotide.

114. The oligo- or polynucleotide of claim 87, wherein said oligo- or polynucleotide comprises an oligo- or polyribonucleotide.

115. The oligo- or polynucleotide of claim 87, wherein said oligo- or polynucleotide comprises an oligo- or polyribonucleotide and further comprises at least one deoxyribonucleotide.

116. The oligo- or polynucleotide of claim 87, having the structural formula:

wherein m and n represent integers from 0 up to about 100,000, and wherein said Sig moiety is attached to at least one of the phosphate moieties in said structural formula.