Universal methylation profiling methods

• US 10,337,049 B2
• Filed: 12/17/2015
• Issued: 07/02/2019
• Est. Priority Date: 06/17/2013
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Bestor, Timothy H., Ju, Jingyue, Li, Xiaoxu, Russo, James J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/973,637
• Publication Number:
  US 20160355542A1
• Time in Patent Office: 1,293 Days
• Field of Search: 435 61
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/167 with ribosyl as the saccharide radical
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/101 radioactivity, e.g. radioactive labels
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C12Y 201/01 Methyltransferases (2.1.1)

Abstract

The present invention provides a method of determining whether cytosine residues present at a predetermined positions within a single strand of a double stranded DNA of known sequence are methylated as well as compounds for carrying out this method.

Claims

1. A method of determining whether a cytosine present at a predefined position within a single strand of a double-stranded DNA of known sequence is non-methylated comprising:

a) contacting the double-stranded DNA with a CpG methyltransferase and an S-adenosylmethionine analog having the structure;

2. The method of claim 1, wherein the chemical group R has any one of the following structures:

3. The method of claim 1, wherein the CpG methyltransferase is M.SssI methyltransferase or a mutant thereof having methyltransferase activity, M.HhaI methyltransferase or a mutant thereof having methyltransferase activity, or M.CviJI methyltransferase or a mutant thereof having methyltransferase activity.

4. The method of claim 1, wherein the cytosine at the predetermined position in the single strand of the double-stranded DNA is present in a CpG site.

5. The method of claim 1, wherein in step d) the determination of the sequence is obtained by sequencing by synthesis.

6. The method of claim 1, further comprising attaching the single strand from the photoirradiated derivative of the double-stranded DNA to a solid support after step c) and prior to step d).

7. The method of claim 1, wherein the CpG methyltransferase is the mutant M.SssI methyltransferase comprising relative to non-mutant M.SssI methyltransferase from E. coli K12 strain ER1821 (i) substitution of serine for the amino acid Q142 and (ii) substitution of serine for the amino acid N370.

8. The method of claim 1, wherein the photoirradiation in step b) is carried out in the presence of a catalyst having long wavelength absorption properties.

9. The method of claim 8, wherein the catalyst is thioxanthone (TX) or a derivative thereof having catalytic properties.

10. The method of claim 1, wherein the photoirradiation in step b) is carried out at a temperature between 0°

C. and 90°

C.

11. The method of claim 1, wherein the photoirradiation in step b) is carried out in a buffered solution with pH between 4 and 10.

12. The method of claim 8, wherein the catalyst is covalently linked to the S-adenosylmethionine analog.

13. A method of determining whether a cytosine present at a predefined position within a single strand of a double-stranded DNA of known sequence is non-methylated comprising:

a) contacting the double-stranded DNA with a CpG methyltransferase and an S-adenosylmethionine analog having the structure;

14. The method of claim 13, wherein the CpG methyltransferase is the mutant M.SssI methyltransferase comprising relative to non-mutant M.SssI methyltransferase from E. coli K12 strain ER1821 (i) substitution of serine for the amino acid Q142 and (ii) substitution of serine for the amino acid N370.