Methods and compositions for labeling nucleic acids

• US 20070207476A1
• Filed: 10/27/2006
• Published: 09/06/2007
• Est. Priority Date: 10/27/2005
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Salic, Adrian, Mitchison, Timothy
• Granted Patent: US 7,910,335 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The present invention relates to methods for the labeling of nucleic acid polymers in vitro and in vivo. In particular, the methods include a [3+2] cycloaddition between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent attached to a label. Such methods do not require fixation and denaturation and therefore can be applied to the labeling of nucleic acid polymers in living cells and in organisms. Also provided are methods for measuring cellular proliferation. In these methods, the amount of label incorporated into the DNA is measured as an indication of cellular proliferation. The methods of the invention can be used in a wide variety of applications including clinical diagnosis of diseases and disorders in which cellular proliferation is involved, toxicity assays, and as a tool for the study of chromosomes'"'"' ultrastructures.

Claims

1. A method of labeling a nucleic acid polymer, comprising steps of:

providing a nucleic acid polymer comprising at least one nucleotide analogue that comprises a first reactive unsaturated group; and

contacting the nucleic acid polymer with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second unsaturated groups.

2. The method of claim 1, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.

3. The method of claim 2, wherein the 1,3-dipole comprises an azide group and the dipolarophile comprises an ethynyl bond.

4. The method of claim 1, wherein the label is directly detectable.

5. The method of claim 4, wherein the label comprises a fluorescent agent.

6. The method of claim 1, wherein the label is indirectly detectable.

7. The method of claim 6, wherein the label comprises a hapten.

8. The method of claim 1, wherein the nucleic acid polymer is inside a cell or in an organism.

9. The method of claim 8, wherein the at least one nucleotide analogue is incorporated into the nucleic acid polymer during DNA replication.

10. The method of claim 1, wherein the step of contacting is performed under aqueous conditions in presence of Cu(I).

11. The method of claim 8, wherein the step of contacting is performed under aqueous conditions in absence of Cu(I) and the reagent further comprises a Cu chelating moiety.

41. A nucleic acid polymer comprising at least one nucleotide analogue attached to a label, wherein said nucleic acid polymer is prepared by the method of claim 1.

42. The nucleic acid polymer of claim 41, wherein the at least one nucleotide analogue comprises a cycloadduct resulting from a [3+2] cycloaddition between an ethynyl group and an azide group.

43. The nucleic acid polymer of claim 41, wherein the label is directly detectable.

44. The nucleic acid polymer of claim 43, wherein the label comprises a fluorescent agent.

45. The nucleic acid polymer of claim 41, wherein the label is indirectly detectable.

46. The nucleic acid polymer of claim 45, wherein the label comprises a hapten.

53. A cell comprising a nucleic acid polymer according to claim 42 or 47.

12. A method of dually labeling a nucleic acid polymer, comprising steps of:

providing a nucleic acid polymer comprising at least one first nucleotide analogue that comprises a first reactive unsaturated group and at least one second nucleotide analogue that comprises a second reactive unsaturated group;

contacting the nucleic acid polymer with a first reagent comprising a third reactive unsaturated group attached to a first label, such that a [3+2] cycloaddition occurs between the first and third unsaturated groups; and

contacting the nucleic acid polymer with a second reagent comprising a fourth reactive unsaturated group attached to a second label, such that a [3+2] cycloaddition occurs between the second and fourth unsaturated groups.

13. The method of claim 12, wherein the first reactive unsaturated group comprises a first 1,3-dipole and the third reactive unsaturated group comprises a first dipolarophile, and wherein the second reactive unsaturated group comprises a second dipolarophile and the fourth reactive unsaturated group comprises a second 1,3-dipole.

14. The method of claim 12, wherein the first reactive unsaturated group comprises a first dipolarophile and the third reactive unsaturated group comprises a first 1,3-dipole, and wherein the second reactive unsaturated group comprises a second 1,3-dipole and the fourth reactive unsaturated group comprises a second dipolarophile.

15. The method of claim 13 or 14, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group and wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group.

16. The method of claim 12, wherein the first and second labels are directly detectable.

17. The method of claim 16, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent, and the first and second fluorescent agents produce a dual-color fluorescence upon excitation.

18. The method of claim 12, wherein the first and second labels are indirectly detectable.

19. The method of claim 18, wherein the first label comprises a first hapten and the second label comprises a second hapten.

20. The method of claim 12, wherein the nucleic acid polymer is inside a cell or in an organism.

21. The method of claim 20, wherein the at least one first nucleotide analogue and at least second one nucleotide analogue are incorporated into the nucleic acid polymer during DNA replication.

22. The method of claim 12, wherein the steps of contacting are performed simultaneously.

23. The method of claim 12, wherein the steps of contacting are performed sequentially.

24. The method of claim 12, wherein the steps of contacting are performed under aqueous conditions in presence of Cu(I).

25. The method of claim 20, wherein the steps of contacting are performed under aqueous conditions in absence of Cu(I) and the reagent further comprises a Cu chelating moiety.

34. The method of claim 25, wherein the first nucleic acid polymer is inside a first cell and the second nucleic acid polymer is inside a second cell.

36. The method of claim 34 or 35, wherein the at least one first nucleotide analogue is incorporated into the first nucleic acid polymer during DNA replication, and the at least one second nucleotide analogue is incorporated into the second nucleic acid polymer during DNA replication.

40. The method of claim 34 or 35, wherein the steps of contacting are performed under aqueous conditions in absence of Cu(I) and the first reagent further comprises a first Cu chelating moiety and the second reagent further comprises a second Cu chelating moiety.

47. A nucleic acid polymer comprising at least one first nucleotide analogue attached to a first label and at least one second nucleotide analogue attached to a second label, wherein said nucleic acid polymer is prepared by the method of claim 12.

48. The nucleic acid polymer of claim 47, wherein the at least one first nucleotide analogue comprises a first cycloadduct resulting from a [3+2] cycloaddition between a first ethynyl group and a first azide group and the at least one second nucleotide analogue comprises a second cycloadduct resulting from a [3+2] cycloaddition between a second ethynyl group and an second azide group.

49. The nucleic acid polymer of claim 47, wherein the first and second labels are directly detectable.

50. The nucleic acid polymer of claim 49, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent, and the first and second fluorescent agent produce a dual fluorescence upon excitation.

51. The nucleic acid polymer of claim 47, wherein the first and second label are indirectly detectable.

52. The nucleic acid polymer of claim 51, wherein the first label comprises a first hapten, and the second label comprises a second hapten.

26. A method of differentially labeling nucleic acid polymers, comprising steps of:

providing a first nucleic acid polymer comprising at least one first nucleotide analogue that comprises a first reactive unsaturated group;

providing a second nucleic acid polymer comprising at least one second nucleotide analogue that comprises a second reactive unsaturated group;

contacting the first nucleic acid polymer with a first reagent comprising a third reactive unsaturated group attached to a first label, such that a [3+2] cycloaddition occurs between the first and third unsaturated groups; and

contacting the second nucleic acid polymer with a second reagent comprising a fourth reactive unsaturated group attached to a second label, such that a [3+2] cycloaddition occurs between the second and fourth unsaturated groups.

27. The method of claim 26, wherein the first reactive unsaturated group comprises a first 1,3-dipole and the third reactive unsaturated group comprises a first dipolarophile, and the second reactive unsaturated group comprises a second dipolarophile and the fourth reactive unsaturated group comprises a second 1,3-dipole.

28. The method of claim 26, wherein the first reactive unsaturated group comprises a first dipolarophile and the third reactive unsaturated group comprises a first 1,3-dipole, and the second reactive unsaturated group comprises a second 1,3-dipole and the fourth reactive unsaturated group comprises a second dipolarophile.

29. The method of claim 27 or 28, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group and wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group.

30. The method of claim 26, wherein the first and second labels are directly detectable.

31. The method of claim 30, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent, and the first and second fluorescent agents produce a dual-color fluorescence upon excitation.

32. The method of claim 26, wherein the first and second labels are indirectly detectable.

33. The method of claim 32, wherein the first label comprises a first hapten and the second label comprises a second hapten.

35. The method of claim 26, wherein the first nucleic acid polymer is inside a first organism and the second nucleic acid polymer is inside a second organism.

37. The method of claim 26, wherein the steps of contacting are performed simultaneously.

38. The method of claim 26, wherein the steps of contacting are performed sequentially.

39. The method of claim 35, wherein the steps of contacting are performed under aqueous conditions in presence of Cu(I).

54. A kit for labeling a nucleic acid polymer comprising:

at least one nucleoside analogue that comprises a first reactive unsaturated group; and

a reagent comprising a second reactive unsaturated group attached to a label.

55. The kit of claim 54, wherein the first reactive unsaturated group comprises a 1,3-dipole, the second reactive unsaturated group comprises a dipolarophile and the first and second reactive unsaturated groups can react via [3+2] cycloaddition.

56. The kit of claim 54, wherein the first reactive unsaturated group comprises a dipolarophile, the second reactive unsaturated group comprises α

1,3-dipole, and the first and second reactive unsaturated groups can react via [3+2] cycloaddition.

57. The kit of claim 55 or 56, wherein the 1,3-dipole comprises an azide group and the dipolarophile comprises an ethynyl group.

58. The kit of claim 54, wherein the label is directly detectable.

59. The kit of claim 58, wherein the label comprises a fluorescent agent.

60. The kit of claim 54, wherein the label is indirectly detectable.

61. The kit of claim 60, wherein the label comprises a hapten.

62. The kit of claim 54 further comprising an aqueous medium and Cu(I).

63. A kit for dually labeling a nucleic acid polymer, comprising

at least one first nucleoside analogue that comprises a first reactive unsaturated group;

at least one second nucleoside analogue that comprises a second reactive unsaturated group;

a first reagent comprising a third reactive unsaturated group attached to a first label; and

a second reagent comprising a fourth reactive unsaturated group attached to a second label.

64. The kit of claim 63, wherein the first reactive unsaturated group comprises a first 1,3-dipole, the third reactive unsaturated group comprises a first dipolarophile, and the first and third reactive unsaturated groups can react via [3+2] cycloaddition;

and wherein the second reactive unsaturated group comprises a second dipolarophile, the fourth reactive unsaturated group comprises a second 1,3-dipole, and the second and fourth reactive unsaturated groups can react via [3+2] cycloaddition.

65. The kit of claim 63, wherein the first reactive unsaturated group comprises a first dipolarophile, the third reactive unsaturated group comprises a first 1,3-dipole, and the first and third reactive unsaturated groups can react via [3+2] cycloaddition;

and wherein the second reactive unsaturated group comprises a second dipolarophile, the fourth reactive unsaturated group comprises a second 1,3-dipole, and the second and fourth reactive unsaturated groups can react via [3+2] cycloaddition.

66. The kit of claim 64 or 65, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group or wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group.

67. The kit of claim 63, wherein the first and second label are directly detectable.

68. The kit of claim 67, wherein the first label comprises a first fluorescent agent, the second label comprises a second fluorescent agent and the first and second fluorescent agent produce a dual fluorescence upon excitation.

69. The kit of claim 63, wherein the first and second labels are indirectly detectable.

70. The kit of claim 69, wherein the first label comprises a first hapten and the second label comprises a second hapten.

71. The kit of claim 63 further comprising an aqueous medium and comprising Cu(I).

72. A method of measuring cellular proliferation, comprising steps of:

contacting a cell with an effective amount of a nucleoside analogue that comprises a first reactive unsaturated group such that the nucleoside analogue is incorporated into DNA of the cell;

contacting the cell with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second reactive unsaturated groups; and

determining an amount of label incorporated into the DNA to measure cellular proliferation.

74. The method of claim 72 or 73, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.

75. The method of claim 74, wherein the 1,3-dipole comprises an azide group and the dipolarophile is an ethynyl group.

76. The method of claim 72 or 73, wherein the label is directly detectable.

77. The method of claim 76, wherein the label comprises a fluorescent agent.

78. The method of claim 72 or 73, wherein the label is indirectly detectable.

79. The method of claim 78, wherein the label comprises a hapten.

80. The method of claim 72 or 73, wherein the step of contacting is performed in absence of Cu(I), and the reagent further comprises a Cu chelating moiety.

81. The method of claim 72, wherein the cell is in a multi-well plate.

73. A method of measuring cellular proliferation in an organism, comprising steps of:

administering to an organism an effective amount of a nucleoside analogue that comprises a first reactive unsaturated group such that the nucleoside analogue is incorporated into DNA of cells of the organism;

contacting at least one cell of the organism with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second reactive unsaturated groups; and

determining an amount of label incorporated into the DNA to measure cellular proliferation in the organism.

82. A method for identifying an agent that perturbs cellular proliferation, comprising steps of:

(a) contacting a cell with a test agent;

(b) contacting the cell with an effective amount of a nucleoside analogue that comprises a first reactive unsaturated group such that the nucleoside analogue is incorporated into DNA of the cell;

(c) contacting the cell with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second reactive unsaturated groups;

(d) determining an amount of label incorporated into the DNA, wherein the amount of label indicates the extent of cellular proliferation; and

(e) identifying the test agent as an agent that perturbs cellular proliferation if the amount of label measured in step (d) is less than or greater than the amount of label measured in a control application in which the cell is not contacted with the test agent.

83. The method of claim 82, wherein step (b) is performed before step (a).

84. The method of claim 82 further comprising the step of identifying the test agent as an agent that induces cellular proliferation if the amount of label measured in step (d) is greater than the amount of label measured in the control application.

85. The method of claim 82 further comprising the step of identifying the test agent as an agent that inhibits cellular proliferation if the amount of label measured in step (d) is less than the amount of label measured in the control application.

86. The method of claim 82, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.

87. The method of claim 86, wherein the 1,3-dipole comprises an azide group and the dipolarophile is an ethynyl group.

88. The method of claim 82, wherein the label is directly detectable.

89. The method of claim 88, wherein the label comprises a fluorescent agent.

90. The method of claim 82, wherein the label is indirectly detectable.

91. The method of claim 90, wherein the label comprises a hapten.

92. The method of claim 82, wherein the step of contacting is performed in absence of Cu(I) and the reagent further comprises a Cu chelating moiety.

93. The method of claim 82, wherein the cell is in a multi-well plate.

94. A method for identifying an agent that perturbs cellular proliferation in an organism, comprising steps of:

(a) exposing an organism to a test agent;

(b) administering to the organism an effective amount of a nucleoside analogue that comprises a first reactive unsaturated group such that the nucleoside analogue is incorporated into DNA of cells of the organism;

(c) contacting at least one cell of the organism with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second reactive unsaturated groups;

(d) determining an amount of label incorporated into the DNA, wherein the amount of label indicates the extent of cellular proliferation; and

(e) identifying the test agent as an agent that perturbs cellular proliferation in the organism if the amount of label measured in step (d) is less than or greater than the amount of label measured in a control application in which the organism is not exposed to the candidate agent.

95. The method of claim 94, wherein step (b) is performed before step (a).

96. The method of claim 94 further comprising the step of identifying the test agent as an agent that induces cellular proliferation in the organism if the amount of label measured in step (d) is greater than the amount of label measured in the control application.

97. The method of claim 94 further comprising the step of identifying the test agent as an agent that inhibits cellular proliferation in the organism if the amount of label measured in step (d) is less than the amount of label measured in the control application.

98. The method of claim 94, wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole.

99. The method of claim 98, wherein the 1,3-dipole comprises an azide group and the dipolarophile is an ethynyl group.

100. The method of claim 94, wherein the label is directly detectable.

101. The method of claim 100 wherein the label comprises a fluorescent agent.

102. The method of claim 94, wherein the label is indirectly detectable.

103. The method of claim 102, wherein the label comprises a hapten.

104. The method of claim 94, wherein the step of contacting with a reagent is performed in vitro.