Methods and compositions for labeling nucleic acids

• US 10,550,422 B2
• Filed: 10/24/2016
• Issued: 02/04/2020
• 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: Gee, Kyle R., Agnew, Brian, Salic, Adrian, Mitchison, Timothy J.
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US15/332,304
• Publication Number:
  US 20170137866A1
• Time in Patent Office: 1,198 Days
• Field of Search: None
• 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 nucleic acid polymer comprising at least one nucleotide analogue attached to a label, wherein the nucleic acid polymer is prepared by a method of labeling a nucleic acid polymer, comprising steps of:

providing a nucleic acid polymer comprising at least one nucleotide analogue that comprises an azide group; and

contacting the nucleic acid polymer with a reagent comprising a label attached to a substituted triarylphosphine, such that a Staudinger ligation occurs between the azide and substituted triarylphosphine;

wherein the nucleic acid polymer is one of the two DNA molecules of a chromosome.

2. A cell comprising a nucleic acid polymer of claim 1.

3. The nucleic acid polymer of claim 1, wherein the nucleic acid polymer is in a living cell.

4. The nucleic acid polymer of claim 1, wherein the nucleic acid polymer is prepared by a method of labeling a nucleic acid polymer, comprising steps of:

providing a nucleic acid polymer comprising at least one nucleotide analogue that comprises an azide group; and

contacting the nucleic acid polymer with a reagent comprising a label attached to a substituted triarylphosphine, such that a Staudinger ligation occurs between the azide and substituted triarylphosphine,

wherein the azide group is attached to the nucleotide analogue at a 5-position of a pyrimidine base.

5. A cell comprising the nucleic acid polymer of claim 4.

6. The nucleic acid polymer of claim 4, wherein the nucleic acid polymer is in a living cell.

7. The nucleic acid polymer of claim 1, wherein the nucleic acid polymer is prepared by a method of labeling a nucleic acid polymer, comprising steps of:

providing a nucleic acid polymer comprising at least one nucleotide analogue that comprises an azide group; and

contacting the nucleic acid polymer with a reagent comprising a label attached to a substituted triarylphosphine, such that a Staudinger ligation occurs between the azide and substituted triarylphosphine,

wherein the azide group is carried on a 5-azido-2′

-deoxyuracil unit.

8. A cell comprising the nucleic acid polymer of claim 7.

9. The nucleic acid polymer of claim 7, wherein the nucleic acid polymer is in a living cell.