SELECTIVE OXIDATION OF 5-METHYLCYTOSINE BY TET-FAMILY PROTEINS

• US 20170145484A1
• Filed: 06/27/2016
• Published: 05/25/2017
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation, United States Department Of Health And Human Services
• Original Assignee: Children's Medical Center Corporation, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Rao, Anjana, Tahiliani, Mamta, Koh, Kian Peng, Agarwal, Suneet, Iyer, Aravind
• Granted Patent: US 10,443,091 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2501/724 Glycosyltransferases (EC 2.4.)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2522/10 Nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.

Claims

1. A kit for modulating gene transcription via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine, the kit comprising the following separate components:

(a) at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, or nucleic acid molecule that comprises a sequence encoding at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment, or combination thereof in an appropriate buffer or solution; and

(b) packaging materials and instructions therein to use said kit to hydroxylate 5-methylcytosine to 5-hydroxymethylcytosine, for the purpose of modulating gene transcription.

2. The kit of claim 1, further comprising at least one reagent suitable for the detection of 5-hydroxymethylcyto sine.

3. The kit of claim 2, wherein the reagent suitable for the detection of 5-hydroxymethylcytosine is an antibody, an antigen-binding portion thereof, an intrabody, or a protein, that specifically binds to 5-hydroxymethylcytosine.

4. The kit of claim 3, wherein the reagent suitable for the detection of 5-hydroxymethylcytosine is an antibody, an antigen-binding portion thereof, an intrabody, or a protein that is specific for cytosine-5-methylsulfonate.

5. A kit for the detection and purification of methylcytosine and 5-hydroxymethylcytosine for use in downstream applications, the kit comprising:

(a) one or more catalytically active TET family enzymes, functional TET family derivatives, or TET catalytically active fragments thereof for the conversion of methylcytosine to 5-hydroxymethylcytosine;

(b) one or more enzymes encoded by bacteriophages of the “

T even”

family;

(c) one or more glucose or glucose-derivative donor substrates;

(d) one or more proteins to detect glucose or glucose-derivative modified nucleotides;

(e) standard DNA purification columns, buffers, and substrate solutions; and

(f) packaging materials and instructions therein to use said kits.

6. The kit of claim 5, wherein the enzyme encoded by bacteriophages of the “

T even”

family is selected from the group consisting of alpha-glucosyltransferases, beta-glucosyltransferases, and beta-glucosyl-alpha-glucosyl-transferases.

7. The kit of claim 6, wherein the alpha-glucosyltransferase is encoded by a bacteriophage selected from the group consisting of T2, T4, and T6 bacteriophages.

8. The kit of claim 6, wherein the beta-glucosyltransferase is encoded by a bacteriophage selected from T4 bacteriophages.

9. The kit of claim 6, wherein the beta-glucosyl-alpha-glucosyl-transferase is encoded by a bacteriophage selected from the group consisting of T2 and T6 bacteriophages.

10. The kit of claim 5, wherein the glucose or glucose-derivative donor substrate is uridine diphosphate glucose (UDPG).

11. The kit of claim 5, wherein the glucose or glucose-derivative donor substrate is radiolabeled.

12. The kit of claim 11, wherein the uridine diphosphate glucose is radiolabeled with 14C or 3H.

13. The kit of claim 5, wherein the protein to detect glucose or glucose-derivative modified nucleotides is a lectin, an antibody or an antigen binding fragment thereof, or an enzyme.

14. The kit of claim 13, wherein the protein recognizes the glucose or glucose-derivative only in the context of 5-hydroxymethylcytosine.

15. The kit of claim 13, wherein the antibody or antigen binding fragment thereof is modified with at least one tag.

16. The kit of claim 15, wherein the tag is a biotin molecule, a bead, a gold particle, or a fluorescent molecule.

17. The kit of claim 13, wherein the enzyme is a hexokinase or a beta-glucosyl-alpha-glucosyl-transferase.

18. The kit of claim 13, wherein the lectin is Musa acuminata lectin (BanLec).

19. The kit of claim 13, wherein the lectin is modified with a gold particle or a fluorescent tag.

20. A method for detecting 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting a nucleic acid with an enzyme and utilizing glucose or glucose-derivative donor substrates that trap covalent enzyme-DNA intermediates to detect 5-hydroxymethylcytosine residues, wherein the enzyme is an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase.

21. The method of claim 20, wherein the glucose donor substrate is a uridine diphosphate glucose analog.

22. The method of claim 21, wherein the uridine diphosphate glucose analog is uridine-2-deoxy-2-fluoro-glucose.

23. The method of claim 20, wherein the 5-hydroxymethylcytosine is naturally occurring.

24. The method of claim 20, further comprising the step of first contacting said nucleic acid with at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, thereby converting 5-methylcytosine to 5-hydroxymethylcytosine.

25. The method of claim 20, wherein the enzyme is tagged.

26. The method of claim 20, in which the alpha-glucosyltransferase is encoded by a bacteriophage selected from the group consisting of T2, T4, and T6 bacteriophages.

27. The method of claim 26, in which the beta-glucosyltransferase is encoded by a bacteriophage selected from T4 bacteriophages.

28. The method of claim 26, in which the beta-glucosyl-alpha-glucosyl-transferase is encoded by a bacteriophage selected from the group consisting of T2 and T6 bacteriophages.

29. The method of claim 20, wherein the nucleic acid is contacted in vitro, in a cell, or in vivo.