Methods and compositions for labeling nucleic acids

• US 9,512,465 B2
• Filed: 08/21/2013
• Issued: 12/06/2016
• 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), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Gee, Kyle R., Agnew, Brian, Salic, Adrian, Mitchison, Timothy J.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/972,365
• Publication Number:
  US 20140065605A1
• Time in Patent Office: 1,203 Days
• Field of Search: None
• US Class Current: 1/1
• 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. Certain methods are provided that include a [3+2] cycloaddition between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent attached to a label. Other methods are provided that include a Staudinger ligation between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent comprising a substituted triarylphosphine 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 differentially labeling DNA, comprising steps of:

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

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

contacting the first cell with a first reagent comprising a first label attached to a first substituted triarylphoshine, such that a Staudinger ligation occurs between the first azide and first substituted triarylphosphine; and

contacting the second cell with a second reagent comprising a second label attached to a second substituted triarylphosphine, such that a Staudinger ligation occurs between the second azide and second substituted triarylphosphine.

2. The method of claim 1, wherein one of the aryl groups of the first triarylphosphine is substituted with a alkyl ester and one of the aryl groups of the second triarylphosphine is substituted with a alkyl ester.

3. The method of claim 2, wherein the alkyl ester on the first triarylphosphine is in ortho to the phosphorus atom of the first triarylphosphine and the alkyl ester on the second triarylphosphine is in ortho to the phosphorus atom of the second triarylphoshine.

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

5. The method of claim 2, 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.

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

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

8. A method of differentially labeling DNA, comprising steps of:

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

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

contacting at least one cell of the first organism with a first reagent comprising a first label attached to a first substituted triarylphoshine, such that a Staudinger ligation occurs between the first azide and first substituted triarylphosphine; and

contacting at least one cell of the second organism with a second reagent comprising a second label attached to a second substituted triarylphosphine, such that a Staudinger ligation occurs between the second azide and second substituted triarylphosphine.

9. The method of claim 8, wherein one of the aryl groups of the first triarylphosphine is substituted with a alkyl ester and one of the aryl groups of the second triarylphosphine is substituted with a alkyl ester.

10. The method of claim 9, wherein the alkyl ester on the first triarylphosphine is in ortho to the phosphorus atom of the first triarylphosphine and the alkyl ester on the second triarylphosphine is in ortho to the phosphorus atom of the second triarylphoshine.

11. The method of claim 8, wherein the first and second labels are directly detectable.

12. The method of claim 9, 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.

13. The method of claim 8, wherein the first and second labels are indirectly detectable.

14. The method of claim 13, wherein the first label comprises a first hapten and the second label comprises a second hapten.