METHODS OF DETECTING 5-HYDROXYMETHYLCYTOSINE AND DIAGNOSING OF CANCER

• US 20180327855A1
• Filed: 11/10/2016
• Published: 11/15/2018
• Est. Priority Date: 11/11/2015
• Status: Active Application

• Current Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Original Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Inventors: EBENSTEIN, Yuval, ZIRKIN, Shahar, MICHAELI HOCH, Yael
• Application Number:
  US15/775,015
• Publication Number:
  US 20180327855A1
• Time in Patent Office: Days
• 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 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2600/154 Methylation markers

Abstract

A method of detecting the level of 5-hydroxymethylcytosines (5-hmc) in a DNA molecule of a cell having a 5-hmc prevalence lower than 0.002% of total DNA bases is provided. The method comprising:

• • (a) attaching a 5-hmc labeling agent to the DNA molecule; and
  • (b) subjecting the DNA molecule to an imaging method suitable for detecting the labeling agent, thereby detecting the level of 5-hmc in the DNA molecule. Also provided is a method of diagnosing cancer in a subject in need thereof, the method comprising:
  • (a) providing a DNA sample of a cell of the subject;
  • (b) detecting the level of 5-hmc in the DNA sample as described herein;
    wherein a significant decrease in the level of 5-hmc in the DNA sample, as compared to a control DNA sample from a healthy subject is indicative that the subject has cancer.

Claims

1. A method of detecting the level of 5-hydroxymethylcytosines (5-hmc) in a DNA molecule of a cell having a 5-hmc prevalence lower than 0.0019% of total DNA bases, the method comprising:

(a) attaching a 5-hmc labeling agent to the DNA molecule; and

(b) subjecting the DNA molecule to an imaging method suitable for detecting said labeling agent, thereby detecting the level of 5-hmc in the DNA molecule.

2. The method of claim 1, wherein said cell is a cancer cell.

3. The method of claim 1, wherein said cell is a cell line.

4. The method of claim 1, wherein said cell is a primary cell.

12. The method of claim 1, wherein said cancer is a soft tissue cancer.

13. The method of claim 12, wherein said soft tissue cancer is selected from the group consisting of leukemia and multiple myeloma.

14. The method of claim 1, wherein said cancer is a solid tumor.

15. The method of claim 14, wherein said cancer is of the gastrointestinal system (GI).

20. The method of claim 1, wherein attaching said labeling agent comprises:

reacting a labeling agent derivatized by a second reactive group with a DNA molecule in said DNA sample in which the 5-hydroxymethylcytosines are glycosylated by a glucose molecule derivatized by a first reactive group,

wherein said first and second reactive groups are chemically compatible to one another.

21. The method of claim 20, wherein glycosylating the 5-hydroxymethylcytosines in the DNA molecule comprises incubating the DNA molecule with β

-glucosyltransferase and a uridine diphosphoglucose (UDP-Glu) derivatized by said first reactive group.

22. The method of claim 20, wherein one of said first and second reactive groups is azide and the other is alkyne, such that attaching said labeling agent to said DNA molecule is effected by a click chemistry.

23. The method of claim 20, wherein said reacting is free of a copper catalyst.

24. The method of claim 20, wherein said first reactive group is azide.

25. The method of claim 24, wherein said uridine diphosphoglucose (UDP-Glu) derivatized by said first reactive group is a UDP-6-N₃-Glucose.

26. The method of claim 25, wherein said UDP-6-N₃-Glucose is synthesized chemically or enzymatically.

28. The method of claim 1, wherein said labeling agent is a fluorescent labeling agent.

29. The method of claim 1, further comprising extending DNA molecule(s) of said DNA sample prior to imaging.

31. The method of claim 29, wherein said extending is effected following step (a).

32. The method of claim 29 not comprising subjecting the DNA molecule(s) to fragmentation.

34. The method of claim 29 further comprising identifying a position of said 5-hydroxymethyl-cytosine (5-hmc) along said DNA molecule(s).

5. A method of diagnosing cancer or pre-malignant lesion in a subject in need thereof, the method comprising:

(a) providing a DNA sample of a cell of the subject;

(b) detecting the level of 5-hmc in the DNA sample by;

(i) attaching a 5-hmc labeling agent to a DNA molecule in the DNA sample; and

(ii) subjecting the DNA molecule to an imaging method suitable for detecting said labeling agent, thereby detecting the level of 5-hmc in the DNA molecule of said DNA sample;

wherein a significant decrease in the level of 5-hmc in the DNA sample, as compared to a control DNA sample from a healthy subject is indicative that the subject has cancer or a pre-malignant lesion.

6. The method of claim 5, wherein said significant decrease is below 50%.

11. The method of claim 5, wherein said cell has a 5-hmc prevalence lower than 0.0019%.

17. The method of claim 5, wherein said cancer is a soft tissue tumor or a solid tumor and said cell is a PBMC.

19. The method of claim 5, wherein said cancer is a solid tumor and said cell is of said tumor (in situ or metastasis).

7-10. -10. (canceled)

16. (canceled)

18. (canceled)

27. (canceled)

30. (canceled)

33. (canceled)

35-38. -38. (canceled)