# US 2014/0194299 A1 · Compositions, methods and related uses for cleaving modified dna

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- Number: US 2014/0194299 A1
- Document ID: US20140194299A1
- Status: Published application
- Kind: A1
- Application number: 14/168,825
- Filed: 2014-01-30
- Published: 2014-07-10
- Earliest claimed: 2008-12-23
- Assignees: New England Biolabs, Inc.
- Applicants: New England Biolabs, Inc.
- Inventors: YU ZHENG; RICHARD J. ROBERTS
- Web page: https://patentgrep.com/patent/US20140194299A1

## CPC

- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6869: Methods for sequencing
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Compositions, methods and related uses are provided relating to cleaving modified DNA. For example, a set of DNA fragments obtainable by enzymatic cleavage of a large DNA is described where at least 50% are similarly sized and have a centrally positioned modified nucleotide. In addition, an enzyme preparation is provided that includes one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide. The one or more enzymes are further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X)10YXGD. The related uses include creating a methylome, methods of purifying DNA fragments containing a modified nucleotide and diagnostic applications.

## Claims

### Claim 1

A set of double-stranded oligonucleotide fragments obtainable by enzymatic cleavage of a large DNA, the large DNA containing one or more modified nucleotides, the set comprising fragments wherein at least 50% are of a similar size and have a centrally positioned modified nucleotide.

### Claim 2 (depends on 1)

A set according to claim 1, wherein the one or more of the fragments are isolated from the set.

### Claim 3 (depends on 1)

A set of oligonucleotide fragments according to claim 1, wherein the large DNA is at least 100 nucleotides in length.

### Claim 4 (depends on 1)

A set of oligonucleotide fragments according to claim 1, wherein the large DNA is a mammalian genomic DNA.

### Claim 5 (depends on 1)

A set of oligonucleotide fragments according to claim 1, wherein the large DNA is human genomic DNA.

### Claim 6 (depends on 1)

A set of oligonucleotide fragments according to claim 1, wherein the centrally located modified nucleotide is a cytosine.

### Claim 7 (depends on 6)

A set of oligonucleotide fragments according to claim 6, wherein the centrally located modified cytosine is proximate to a guanine.

### Claim 8 (depends on 7)

A set of oligonucleotide fragments according to claim 7, wherein the modified cytosine is a methylated or hydroxymethylated cytosine.

### Claim 9 (depends on 1)

A set of oligonucleotide fragments according to claim 1, wherein the fragments are less than 60 nucleotides in size.

### Claim 10 (depends on 9)

A set of oligonucleotide fragments according to claim 9, wherein the fragments have a similar size in the range of 28-36 nucleotides.

### Claim 11 (depends on 1)

A set of oligonucleotide fragments according to claim 1, wherein at least one of the modified nucleotides is located within 30 nucleotides from one end of the fragment.

### Claim 12 (depends on 1)

An enzyme preparation, comprising: at least one enzyme that recognizes a modified nucleotide in a DNA and cleaves the DNA at a site that is distant from the modified nucleotide thereby generating a set of fragments as claimed in claim 1, the at least one enzyme further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X)10YXGD.

### Claim 13 (depends on 12)

An enzyme preparation according to claim 12, wherein the at least one enzyme cleaves the DNA at a non-random distance from the modified nucleotide.

### Claim 14 (depends on 12)

An enzyme preparation according to claim 12, wherein the at least one enzyme has an N-terminal conserved domain with greater than 90% sequence homology with WXD(X)6G(X)3YXGD(X)10-15GN(X)2L X10-20PX3F.

### Claim 15 (depends on 12)

An enzyme preparation according to claim 12, wherein the at least one enzyme comprises a recognition domain and a cleavage domain encoded by a single open reading frame.

### Claim 16 (depends on 12)

An enzyme preparation according to claim 12, wherein the at least one enzyme has a C-terminal conserved domain with greater than 90% amino acid sequence homology to FEX20-30DX2-4DX19-22(Q/E)XK.

### Claim 17 (depends on 12)

An enzyme preparation according to claim 12, wherein the at least one enzyme has an amino acid sequence with greater than 90% sequence homology to a protein sequence selected from SEQ ID NOS: 7-22.

### Claim 18 (depends on 12)

An enzyme preparation according to claim 12, wherein the at least one enzyme is fused to an affinity tag.

### Claim 19 (depends on 17)

An enzyme preparation according to claim 17, wherein the affinity tag is selected from the group consisting of a chitin-binding domain, maltose-binding domain and a His tag.

### Claim 20 (depends on 12)

An enzyme preparation according to claim 12, further comprising an activator DNA.

### Claim 21 (depends on 12)

An enzyme preparation according to claim 12, wherein the N-terminal domain is capable of being recognized by an antibody.

### Claim 22 (depends on 21)

An antibody as defined in claim 21.

### Claim 23

An enzyme preparation, comprising: one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide, the one or more enzymes further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology with WXD(X)10YXGD.

### Claim 24 (depends on 1)

A method for obtaining a set of oligonucleotide fragments as claimed in claim 1, comprising:
a. enzymatically cleaving a large DNA containing one or more modified nucleotides; and
b. obtaining the set of oligonucleotide fragments.

### Claim 25 (depends on 24)

A method according to claim 24, further comprising separating the set of oligonucleotide fragments from uncleaved DNA.

### Claim 26 (depends on 25)

A method according to claim 25, further comprising: sequencing from the separated set of fragments at least one fragment in the set of fragments to determine the location of one or more modified nucleotides contained within the at least one fragment.

### Claim 27 (depends on 24)

A method according to claim 24, further comprising: analyzing some target oligonucleotide fragments for the presence and location of one or more modified nucleotides in the large DNA.

### Claim 28 (depends on 24)

A method according to claim 24, further comprising: sequencing substantially all the fragments in the set of oligonucleotide fragments and mapping the sequences onto a genome sequence map to determine the location of modified cytosines.

### Claim 29 (depends on 12)

A method for identifying one or more isolated enzymes in the enzyme preparation according to claim 12, comprising:
a. searching a sequence database using a sequence selected from the group consisting of SEQ ID. NO: 7-22 and variants thereof; and
b. identifying additional sequences having an N-terminal region characterized by a consensus sequence of WXD(X)6G (X)3YXGD(X)10-15GN(X)2L X10-20PX3F.

### Claim 30 (depends on 29)

A method according to claim 29, wherein the identified additional sequences have a C-terminal end comprising a catalytic domain with a consensus sequence of FEX20-30DX2-4DX19-22(Q/E)XK.

### Claim 31 (depends on 12)

A method for isolating from a mixture, DNA fragments containing one or more modified nucleotides, comprising:
a. adding to the mixture an enzyme preparation according to claim 12, wherein the at least one enzyme has been mutated so as to lack enzyme cleavage activity, wherein the mutant enzyme is immobilized on a solid surface; and
b. separating the DNA fragments bound to the immobilized enzyme from the mixture.

### Claim 32 (depends on 12)

A method to determine the location of at least one modified nucleotide in a large DNA, comprising:
a. cleaving a large DNA with an enzyme preparation according to claim 12;
b. obtaining a set of oligonucleotide fragments, each fragment containing at least one modified nucleotide; and
c. determining the location of the at least one modified nucleotide in a sequence map of the large DNA by sequencing one or more oligonucleotides in the set of oligonucleotide cleavage products.

### Claim 33 (depends on 12)

A method of identifying a present or future phenotypic property in a cell preparation or tissue sample from a pattern of modified nucleotides; comprising:
a. cleaving into fragments a large DNA from a cell preparation or tissue sample by means of an enzyme preparation described in claim 12; and
b. comparing a location for modified nucleotides in the fragments with a pattern of modified nucleotides in a control DNA so as to determine a present or future phenotypic property.

### Claim 34 (depends on 33)

A method according to claim 33, wherein (a) further comprises: separating fragments with one or more modified nucleotides from fragments lacking a modified nucleotide by:
(i) contacting the cleavage fragments with a molecule that binds to the fragments containing the one or more modified nucleotides with an immobilized preparation of an affinity-binding protein; or
(ii) size separation.

### Claim 35 (depends on 12, 34)

A method according to claim 34, wherein the affinity-binding protein is derived from an enzyme preparation according to claim 12, wherein the enzyme cleavage activity of the at least one enzyme has been inactivated.

### Claim 36 (depends on 33)

A method according to claim 33, where (a) further comprises: identifying on a methylome or a genome a location for the one or more modified nucleotides in the immobilized cleavage fragments.

### Claim 37

A method for obtaining a purified preparation of fragments containing one or more modified nucleotides, comprising:
a. contacting a mixture of DNA fragments in which one or more of the fragments contain at least one modified nucleotide with an immobilized preparation of an affinity-binding molecule;
b. binding the one or more fragments containing at least one modified nucleotide to the affinity-binding molecule; and
c. obtaining a purified preparation of fragments containing one or more modified nucleotides.

### Claim 38 (depends on 12, 37)

A method according to claim 37, wherein the affinity-binding molecule is an enzyme preparation according to claim 12, wherein the enzyme cleavage activity has been inactivated.

### Claim 39 (depends on 38)

A method according to claim 38, wherein the isolated enzymes in the enzyme preparation are associated with a binding moiety.

### Claim 40 (depends on 12)

A kit, comprising: an enzyme preparation according to claim 12, in a container and instructions for use.

### Claim 41 (depends on 40)

A kit according to claim 40, further comprising an activator molecule.