METHODS AND COMPOSITIONS FOR LABELING NUCLEIC ACIDS

• US 20140295414A1
• Filed: 06/12/2014
• Published: 10/02/2014
• Est. Priority Date: 10/27/2005
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Salic, Adrian, Mitchison, Timothy J.
• Granted Patent: US 9,790,541 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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 a base, wherein the first reactive unsaturated group is directly covalently attached to the base; 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;

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 1, 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.

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

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.

105. The method of claim 1, wherein the nucleic acid polymer is prepared by automated, solid-phase procedure using a phosphoramidite synthesis method, and at least one phosphoramidite used in the phosphoramidite synthesis method is a phosphoramidite form of 5-ethynyl-2′

-deoxyuracil.

108. The method of claim 1, wherein the nucleic acid polymer is prepared by an in vitro extension or amplification method.

109. The method of claim 108, wherein at least one nucleoside analogue used in the in vitro extension or amplification method is a triphosphate form of 5-ethynyl-2′

deoxyuracil or 5-azido-2′

-deoxyuracil.

2. (canceled)

8-9. -9. (canceled)

11-25. -25. (canceled)

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 and a base, wherein the first reactive unsaturated group is directly covalently attached to the base;

providing a second nucleic acid polymer comprising at least one second nucleotide analogue that comprises a second reactive unsaturated group and a base, wherein the second reactive unsaturated group is directly covalently attached to the base;

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;

wherein the first reactive unsaturated group comprises a first 1,3-dipole and the third reactive unsaturated group comprises a first dipolarophile, the second reactive unsaturated group comprises a second dipolarophile and the fourth reactive unsaturated group comprises a second 1,3-dipole, or

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 26, 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.

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 26, wherein the steps of contacting are performed under aqueous conditions in presence of Cu(I).

106. The method of claim 26, wherein the first nucleic acid polymer is prepared by automated, solid-phase procedure using a phosphoramidite synthesis method, and at least one phosphoramidite used in the phosphoramidite synthesis method is a phosphoramidite form of 5-ethynyl-2′

-deoxyuracil.

107. The method of claim 26, wherein the second nucleic acid polymer is prepared by automated, solid-phase procedure using a phosphoramidite synthesis method, and at least one phosphoramidite used in the phosphoramidite synthesis method is a phosphoramidite form of 5-ethynyl-2′

-deoxyuracil.

112. The method of claim 26, wherein the first nucleic acid polymer, second nucleic acid polymer, or both is prepared by an in vitro extension or amplification method.

113. The method of claim 112, wherein at least one nucleoside analogue used in the in vitro extension or amplification method is a triphosphate form of 5-ethynyl-2′

deoxyuracil or 5-azido-2′

-deoxyuracil.

27-28. -28. (canceled)

34-36. -36. (canceled)

40. (canceled)

53. (canceled)

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;

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, or

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

57. The kit of claim 54, 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).

114. The kit of claim 54, wherein the at least one nucleoside analogue is a triphosphate form of 5-ethynyl-2′

deoxyuracil or 5-azido-2′

-deoxyuracil.

115. The kit of claim 54, wherein the at least one nucleoside analogue is a phosphoramidite form of 5-ethynyl-2′

deoxyuracil.

55-56. -56. (canceled)

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;

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, or

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 63, 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).

116. The kit of claim 63, wherein the at least one first nucleoside analogue is a triphosphate form of 5-ethynyl-2′

-deoxyuracil and the at least one second nucleoside analogue is a triphosphate form of 5-azido-2′

-deoxyuracil.

117. The kit of claim 63, wherein the at least one first nucleoside analogue is a triphosphate form of 5-azido-2′

-deoxyuracil and the at least one second nucleoside analogue is a triphosphate form of 5-ethynyl-2′

-deoxyuracil.

118. The kit of claim 63, wherein the at least one first nucleoside analogue is a phosphoramidite form of 5-ethynyl-2′

deoxyuracil.

119. The kit of claim 63, wherein the at least one second nucleoside analogue is a phosphoramidite form of 5-ethynyl-2′

deoxyuracil.

64-65. -65. (canceled)

72-104. -104. (canceled)