Methods and compositions for labeling nucleic acids

• US 7,910,335 B2
• Filed: 10/27/2006
• Issued: 03/22/2011
• 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: President and Fellows of Harvard College (Harvard Corporation), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Mitchison, Timothy J., Salic, Adrian
• Primary Examiner(s): Woolwine; Samuel C
• Application Number:
  US11/588,732
• Publication Number:
  US 20070207476A1
• Time in Patent Office: 1,607 Days
• Field of Search: None
• US Class Current: 435/91.1
• 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 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;

wherein the first reactive unsaturated group comprises an alkynyl group and the second reactive unsaturated group comprises an azide or wherein the first reactive unsaturated group comprises an azide group and the second reactive unsaturated group comprises an alkynyl group.

3. The method of claim 1 or 2, wherein the alkynyl group is an ethynyl group.

4. The method of claim 1 or 2, 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 or 2, wherein the label is indirectly detectable.

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

8. The method of claim 1 or 2, wherein the step of contacting the cell with a reagent is performed in absence of Cu(I), and the reagent further comprises a Cu chelating moiety.

9. The method of claim 1, wherein the cell is in a multi-well plate.

31. The method of claim 1, wherein the first reactive unsaturated group comprises an alkynyl group and the second reactive unsaturated group comprises an azide group.

32. The method of claim 31, wherein the alkynyl group is an ethynyl group.

39. The method of claim 1, wherein the nucleoside analogue is ethynyl-dU.

43. The method of claim 1, wherein the nucleoside analogue is azido-dU.

2. 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;

wherein the first reactive unsaturated group comprises an alkynyl group and the second reactive unsaturated group comprises an azide or wherein the first reactive unsaturated group comprises an azide group and the second reactive unsaturated group comprises an alkynyl group.

33. The method of claim 2, wherein the first reactive unsaturated group comprises an alkynyl group and the second reactive unsaturated group comprises an azide group.

34. The method of claim 33, wherein the alkynyl group is an ethynyl group.

40. The method of claim 2, wherein the nucleoside analogue is ethynyl-dU.

44. The method of claim 2, wherein the nucleoside analogue is azido-dU.

10. 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;

wherein the first reactive unsaturated group comprises an alkynyl group and the second reactive unsaturated group comprises an azide or wherein the first reactive unsaturated group comprises an azide group and the second reactive unsaturated group comprises an alkynyl group.

11. The method of claim 10, wherein step (b) is performed before step (a).

12. The method of claim 10 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.

13. The method of claim 10 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.

14. The method of claim 10, wherein the alkynyl group is an ethynyl group.

15. The method of claim 10, wherein the label is directly detectable.

16. The method of claim 15, wherein the label comprises a fluorescent agent.

17. The method of claim 10, wherein the label is indirectly detectable.

18. The method of claim 17, wherein the label comprises a hapten.

19. The method of claim 10, wherein the step of contacting the cell with a reagent is performed in absence of Cu(I) and the reagent further comprises a Cu chelating moiety.

20. The method of claim 10, wherein the cell is in a multi-well plate.

35. The method of claim 10, wherein the first reactive unsaturated group comprises an alkynyl group and the second reactive unsaturated group comprises an azide group.

36. The method of claim 35, wherein the alkynyl group is an ethynyl group.

41. The method of claim 10, wherein the nucleoside analogue is ethynyl-dU.

45. The method of claim 10, wherein the nucleoside analogue is azido-dU.

21. 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 test agent;

wherein the first reactive unsaturated group comprises an alkynyl group and the second reactive unsaturated group comprises an azide or wherein the first reactive unsaturated group comprises an azide group and the second reactive unsaturated group comprises an alkynyl group.

22. The method of claim 21, wherein step (b) is performed before step (a).

23. The method of claim 21 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.

24. The method of claim 21 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.

25. The method of claim 21, wherein the alkynyl group is an ethynyl group.

26. The method of claim 21, wherein the label is directly detectable.

27. The method of claim 26 wherein the label comprises a fluorescent agent.

28. The method of claim 21, wherein the label is indirectly detectable.

29. The method of claim 28, wherein the label comprises a hapten.

30. The method of claim 21, wherein the step of contacting with a reagent is performed in vitro.

37. The method of claim 21, wherein the first reactive unsaturated group comprises an alkynyl group and the second reactive unsaturated group comprises an azide group.

38. The method of claim 37, wherein the alkynyl group is an ethynyl group.

42. The method of claim 21, wherein the nucleoside analogue is ethynyl-dU.

46. The method of claim 21, wherein the nucleoside analogue is azido-dU.