Methods and compositions for labeling nucleic acids

• US 8,859,753 B2
• Filed: 10/28/2010
• Issued: 10/14/2014
• 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.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/914,338
• Publication Number:
  US 20110065907A1
• Time in Patent Office: 1,447 Days
• Field of Search: None
• US Class Current: 536/25.32
• 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 analog 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 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;

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, the second reactive unsaturated group comprises a second 1,3-dipole and the fourth reactive unsaturated group comprises a second dipolarophile.

2. The method of claim 1, 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.

3. The method of claim 1, wherein the first and second labels are directly detectable.

4. The method of claim 3, 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.

5. The method of claim 1, wherein the first and second labels are indirectly detectable.

6. The method of claim 5, wherein the first label comprises a first hapten and the second label comprises a second hapten.

7. The method of claim 1, wherein the steps of contacting are performed simultaneously.

8. The method of claim 1, wherein the steps of contacting are performed sequentially.

9. The method of claim 1, wherein the steps of contacting are performed under aqueous conditions in presence of Cu(I).

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

11. The method of claim 10, 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.