Modified nucleotides methods and kits

• US 9,206,216 B2
• Filed: 05/29/2012
• Issued: 12/08/2015
• 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, Schultz, Jean-Samuel
• Primary Examiner(s): Olson, Eric
• Application Number:
  US13/482,927
• Publication Number:
  US 20120252691A1
• Time in Patent Office: 1,288 Days
• Field of Search: None
• US Class Current: 1/1
• 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 compound having the structure (II):

2. The compound of claim 1 where the sugar is ribose;

the purine or pyrimidine base is cytosine (C);

m is 3;

Lnk is

3. The compound of claim 1 where the sugar is ribose, and the purine or pyrimidine base is selected from cytosine (C), uracil (U), adenine (A), guanine (G), or inosine (I).

4. The compound of claim 3 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.

5. The compound of claim 1 where the observable label is a chromogen, a fluorophore, a mass label, a spin label, a streptavidin-binding label, or a secondary detection label.

6. The compound of claim 1 where n is an integer selected from 2 to 24 inclusive.

7. The compound of claim 1 where 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.

8. The compound of claim 1 where the sugar is ribose;

the purine or pyrimidine base is cytosine (C);

m is 3;

Lnk is

9. The compound of claim 1 including a salt, conjugate base, tautomer, and/or ionized form of the compound.

10. A method for enriching for an RNA-binding protein or RNA binding complex, the method comprising

labeling an RNA with the compound of claim 1 by incubating under suitable conditions the RNA and the compound of claim 1 with an enzyme capable of ligating the compound of claim 1 to the RNA molecule to result in a labeled RNA,

contacting a protein source that contains at least one RNA-binding protein or RNA binding complex with the labeled RNA under conditions suitable for forming a complex between the labeled RNA and the RNA-binding protein, and

enriching for the RNA-binding protein or RNA binding complex utilizing the labeled RNA as bait.

11. The method of claim 10 where the RNA is labeled by contacting the RNA with an excess of the compound.

12. The method of claim 11 further comprising a step of including a crosslinking agent to the compound.

13. The method of claim 11 where the affinity handle is selected from at least one of biotin-PEG₄-alkane-3′

5′

-bisphosphate cytidine;

desbiotin-PEG₄-alkane-3′

5′

-bisphosphate cytidine, biotin-PEG₁₂-alkane-3′

5′

-bisphosphate cytidine;

desbiotin-PEG₁₂-alkane-3′

5′

-bisphosphate cytidine;

azido-PEG₄-alkane-3′

5′

-bisphosphate cytidine;

azido-PEG₁₂-alkane-3′

5′

-bisphosphate cytidine;

TMT-PEG₄-alkane-3′

5′

-bisphosphate cytidine; and

/or TMT-PEG₁₂-alkane-3′

5′

-bisphosphate cytidine.

14. The method of claim 10 where the protein source is at least one of cell lysate, tissue lysate, exogenously expressed protein in cell or tissue lysates, protein expressed in an in vitro transcription and/or translation system, and/or protein purified form bacteria, yeast, fungi, tissue, or mammalian cells.

15. The method of claim 14 where the labeled RNA is contacted with the protein lysate and affinity resin or matrix in an optimized binding buffer.

16. The method of claim 15 further comprising washing, eluting, and detecting the RNA protein complex.

17. The method of claim 16 where detection is by Western blot, array, and/or mass spectrometry.

18. The method of claim 10 wherein the labeled RNA contains a crosslinker within the ligated compound, the compound attached to the RNA using T4 RNA ligase.

19. The method of claim 10 wherein the labeled RNA contains a crosslinker cleavable within the ligated compound that is attached to the RNA using T4 RNA ligase.

20. The method of claim 10 further comprising a step utilizing a crosslinking reagent.

21. The method of claim 10 using an affinity matrix to enrich for the RNA-binding protein or RNA binding complex, where the affinity matrix is streptavidin agarose resin, streptavidin magnetic breads, hydrazide resin, alkoxyamine resin, modified agarose resin, soft-release streptavidin magnetic beads, soft-release agarose resin, and/or resin coupled with at least one anti-tandem mass tag (TMT) antibody.

22. A kit for enriching an RNA binding protein or RNA binding protein complex, the kit comprising the compound of claim 1 and reagents for labeling an RNA with the compound of claim 1.

23. The kit of claim 22 further comprising at least one control comprising labeled RNA and at least one control comprising unlabeled RNA to assess RNA ligation and/or binding affinity.

24. The kit of claim 22 further comprising the compound of claim 7, and instructions for labeling the RNA, resulting in a labeled RNA with a crosslinker, such that the labeled RNA contains an affinity handle and crosslinking reagent.

25. The kit of claim 22 further comprising reagents for capture and detection of RNA-protein interactions.

26. A compound selected from the group consisting of