# US 9,040,239 B2 · Composition and methods of oxygenation of nucleic acids containing 5-methylpyrimidine

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- Number: US 9,040,239 B2
- Document ID: US9040239B2
- Status: Granted
- Kind: B2
- Application number: 13/827,087
- Filed: 2013-03-14
- Published: 2014-05-08
- Granted: 2015-05-26
- Earliest claimed: 2012-03-15
- Assignees: New England Biolabs, Inc.
- Applicants: New England Biolabs, Inc.
- Inventors: Yu Zheng; Lana Saleh; June Pais; Nan Dai; Richard J. Roberts; Ivan R. Correa, Jr.; Megumu Mabuchi; Romualdas Vaisvila
- Web page: https://patentgrep.com/patent/US9040239B2

## CPC

- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C07H 19/06: Pyrimidine radicals
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07K 14/47: from mammals
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- C12P 19/30: Nucleotides
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/68: involving nucleic acids
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C07K 2319/81: containing a Zn-finger domain for DNA binding
- Y02P 20/52: using catalysts, e.g. selective catalysts
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

5-methylpyrimidine oxygenases and their use in the modification of nucleic acids are described.

## Claims

### Claim 1

A composition comprising:
(i) a buffer comprising Fe(II) and alpha-ketoglutarate; and
(ii) a polypeptide that has 5-methylpyrimidine oxygenase activity and has at least 90% identity to amino acids 154-304 of SEQ ID NO: 2.

### Claim 2 (depends on 1)

A composition according to claim 1, wherein the buffer does not contain ATP.

### Claim 3 (depends on 1)

A composition according to claim 1, wherein the buffer contains ATP.

### Claim 4 (depends on 1)

A composition according to claim 1, wherein the buffer is at a pH in the range of pH 6 to pH 8.

### Claim 5 (depends on 4)

A composition according to claim 4, wherein the buffer is at a pH in the range of pH 6 to pH 7.5.

### Claim 6 (depends on 1)

A composition according to claim 1, further comprising a nucleic acid.

### Claim 7 (depends on 1)

A kit comprising a composition according to claim 1 wherein the buffer and the polypeptide are in separate containers.

### Claim 8 (depends on 7)

A kit according to claim 7, wherein the reaction buffer is at a pH in the range of pH 6 to pH 7.5.

### Claim 9 (depends on 7)

A kit according to claim 7, wherein the reaction buffer contains ATP.

### Claim 10 (depends on 7)

A kit according to claim 7, wherein the reaction buffer does not contain ATP.

### Claim 11 (depends on 7)

A kit according to claim 7, further comprising a nucleic acid.

### Claim 12 (depends on 7)

A kit according to claim 7, further comprising a reducing agent.

### Claim 13 (depends on 7)

A kit according to claim 7, further comprising a β-glycosyltransferase (BGT) and UDP-glucosamine.

### Claim 14 (depends on 7)

A kit according to claim 7, further comprising a β-glycosyltransferase (BGT) and UDP-glucose.

### Claim 15 (depends on 7)

A kit according to claim 7, further comprising a DNA glycosylase.

### Claim 16 (depends on 7)

A kit according to claim 7, further comprising an endonuclease.

### Claim 17 (depends on 1)

A method for differentiating a 5-methylcytosine (5-mC) from 5-hydroxymethylcytosine (5-hmC) in a genome or genome fragment, comprising:
a. reacting the isolated genome or genome fragment containing 5-mC and 5-hmC with UDP-glucose or UDP-glucosamine; a glycosyltransferase for transferring glucose or glucosamine to the 5hmC; and a composition according to claim 1;
b. cleaving the glucosylated template with a modification-dependent endonuclease that recognizes at least one of the modified nucleotides; and
c. differentiating the 5-mC from the 5-hmC by an altered cleavage pattern.

### Claim 18 (depends on 17)

A method according to claim 17, wherein the modification-dependent endonuclease is AbaSI.

### Claim 19 (depends on 1)

A composition according to claim 1, wherein the polypeptide further comprises a binding domain.

### Claim 20 (depends on 19)

A composition according to claim 19, wherein the binding domain is selected from the group consisting of: a His-tag, a maltose-binding protein, a chitin binding domain, and a DNA binding domain.

### Claim 21 (depends on 20)

The composition according to claim 20, wherein the binding domain comprises a zinc finger or transcription activator-like (TAL) effector domain.

### Claim 22 (depends on 1)

The composition of claim 1, wherein the polypeptide is a fusion protein.

### Claim 23 (depends on 1)

The composition of claim 1, wherein the polypeptide has at least 95% identity to amino acids 154-304 of SEQ ID NO: 2.

### Claim 24 (depends on 1)

The composition of claim 1, wherein the polypeptide is identical at amino acids 154-304 of SEQ ID NO: 2.

### Claim 25 (depends on 1)

The composition of claim 1, wherein the polypeptide is at least 90% identical to SEQ ID NO: 2.

### Claim 26 (depends on 1)

The composition of claim 1, wherein the polypeptide is at least 95% identical to SEQ ID NO: 2.

### Claim 27 (depends on 1)

The composition of claim 1, wherein the polypeptide is identical to SEQ ID NO: 2.