Modified nucleotides methods and kits

• US 9,828,633 B1
• Filed: 10/15/2015
• Issued: 11/28/2017
• Est. Priority Date: 04/21/2010
• Status: Active Grant

• Current Assignee: Pierce Biotechnology Incorporated (Perspecta, Inc.)
• Original Assignee: Pierce Biotechnology Incorporated (Perspecta, Inc.)
• Inventors: Etienne, Christopher L., Opperman, Kay K., Kaboord, Barbara J., Meier, Scott D., Schultz, Jean-Samuel
• Primary Examiner(s): Olson, Eric S
• Application Number:
  US14/883,810
• Time in Patent Office: 775 Days
• Field of Search: None
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Y 605/01001 DNA ligase (ATP) (6.5.1.1)
• CPC Class Codes: C12Y 605/01002 DNA ligase (NAD+) (6.5.1.2)
• CPC Class Codes: C12Y 605/01003 RNA ligase (ATP) (6.5.1.3)

Abstract

Modified nucleotides, and methods to modify nucleotides with a moiety or label, such as biotin, that permits their detection and results in a modified nucleotide, and methods of use of the modified nucleotide in quantitative and qualitative assays.

Claims

1. A method for labeling a nucleic acid with a modified nucleotide, the method comprising

contacting a sample containing the nucleic acid with the modified nucleotide to form a mixture;

adding a ligase to form a reaction mixture; and

incubating the reaction mixture under conditions sufficient for the ligase to ligate the modified nucleotide to the nucleic acid, to result in a labeled nucleic acid;

where the modified nucleotide has the structure (II);

2. The method of claim 1, the modified nucleotide wherein the sugar is ribose, and the purine or pyrimidine base is selected from cytosine (C), uracil (U), adenine (A), guanine (G), or inosine (I).

3. The method of claim 2, where the purine or pyridine base is selected from 1-methyladenine, N⁶-methyladenine, N⁶-isopentyladenine, N,N-dimethyladenine, 7-deazaadenine, 2-thiocytosine, 3-methylcytosine, N⁴-acetylcytosine, 2-thiocytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, N₂,N₂-dimethylguanine, 7-deazaguanine, 2-thiouracil, 6-thiopurine, or 2,6-diaminopurine.

4. The method of claim 1, the modified nucleotide wherein the observable label is a chromogen, a fluorophore, a mass label, a spin label, a streptavidin-binding label, or a secondary detection label.

5. The method of claim 1, the modified nucleotide wherein n is an integer selected from 2 to 24 inclusive.

6. The method of claim 1, the modified nucleotide wherein the sugar is ribose;

the purine or pyrimidine base is selected from adenine (A), cytosine (C), guanine (G), uracil U), or inosine (I);

m is 3;

n is 4; and

the observable label is a streptavidin-binding compound selected from biotin, desthiobiotin, or iminobiotin.

7. The method of claim 1, the modified nucleotide wherein the sugar is ribose;

the purine or pyrimidine base is cytosine (C);

m is 3;

Lnk is

8. The method of claim 1, wherein the ligase is T4 ligase.

9. The method of claim 8, wherein the ligase is selected from the group consisting of single stranded T4 ligase, double stranded T4 ligase, thermostable T4 ligase, T4 RNA Ligase 1, T4 RNA Ligase 2, T4 RNA Ligase 2, truncated, and T4 RNA Ligase 2, truncated K227Q.

10. The method of claim 1 wherein prior to the contacting step, the sample containing the nucleic acid is heated to a temperature of about 75°

C. to about 95°

C., and then cooled to a temperature of about 2°

C. to about 10°

C., wherein the sample optionally contains dimethylsulfoxide (DMSO) at a concentration ranging from 0% to about 25%.

11. The method of claim 1, wherein the reaction mixture further comprises PEG having a molecular weight between about 1500 and 24,000 inclusive and at a concentration ranging from 5%^w/v PEG to 20%^w/v PEG inclusive.

12. The method of claim 1, wherein the conditions sufficient for the ligase to ligate the modified nucleotide to the nucleic acid comprise incubating the reaction mixture between 30 minutes and 16 hours at a temperature ranging between 16°

C. and 37°

C.

13. The method of claim 1, wherein the nucleic acid is RNA.

14. A method for detecting a nucleic acid binding biomolecule, the method comprising

forming a labeled nucleic acid by

labeling a nucleic acid with a modified nucleotide, the method comprising contacting a sample containing the nucleic acid with the modified nucleotide to form a mixture;

adding a ligase to form a reaction mixture; and

incubating the reaction mixture under conditions sufficient for the ligase to ligate the modified nucleotide to the nucleic acid, to result in a labeled nucleic acid;

incubating the labeled nucleic acid with a biological sample thought to contain the nucleic acid binding biomolecule to form a complex; and

detecting the complex of the nucleic acid binding biomolecule bound to the labeled nucleic acid;

where the modified nucleotide has the structure (II);

15. The method of claim 14, wherein the biological sample is a purified protein, a tissue sample, whole tissue, whole organ, cell culture, cell extract, cell lysate, or in vitro translated protein lysate.

16. The method of claim 14, wherein the labeled nucleic acid is RNA and the nucleic acid binding biomolecule is an RNA-binding protein.

17. The method of claim 16, wherein the complex of the labeled RNA and the RNA-binding protein is stabilized by crosslinking the RNA-binding protein to the labeled RNA by the modifying molecule of the modified nucleotide or by adding crosslinkers after the labeled RNA/RNA-binding protein complex is formed.

18. The method of claim 14, wherein detection of the complex comprises associating the complex with a solid support, eluting the complex from the solid support, and detecting the eluted complex.

19. The method of claim 18, wherein the solid support is a bead, a plate, or a column.

20. The method of claim 18, wherein the Obs of the modified nucleotide is biotin, desthiobiotin or iminobiotin and the solid support comprises a streptavidin.

21. The method of claim 14, wherein detection comprises a mobility shift assay comprising subjecting the complex to electrophoresis and detecting a shift in molecular weight of the complex compared to the nuclei acid binding biomolecule alone.

22. The method of claim 14, wherein detection comprises subjecting the biological sample to electrophoresis, transferring the electrophoresed biological sample to a membrane, probing the membrane with the labeled nucleic acid, and detecting the complex.

23. The method claim 14, wherein detection comprises in situ hybridization.