Compositions and Methods for Identifying Hydroxymethylcytosine in a DNA

• US 20150004596A1
• Filed: 06/27/2014
• Published: 01/01/2015
• Est. Priority Date: 06/28/2013
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Zhu, Zhenyu, Borgaro, Janine, Guan, Shengxi, Quimby, Aine, Sun, Zhiyi
• Granted Patent: US 10,597,647 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2537/161 A competitive reaction step

Abstract

Provided herein in some embodiments is a non-naturally occurring variant of a wild type restriction enzyme defined by SEQ ID NO: 20, wherein the variant has at least a 2 fold increase in cleavage at 5-β glucosylhydroxymethylcytosine (5βghmC) compared with methylcytosine relative to the wild type enzyme. Methods for examining hydroxymethylation of a DNA sample using the variant enzyme are also provided.

Claims

1. A non-naturally occurring variant of a wild type restriction enzyme wherein the wild type restriction enzyme is defined by SEQ ID NO:

20, and wherein the variant has at least 90% sequence identity to the wild type enzyme and has at least a 2 fold increase in cleavage at 5β

ghmC compared with 5 mC relative to the wild type enzyme.

2. The variant of claim 1, wherein the variant has one or more amino acid substitutions at a position corresponding to V72, T152 or R282 of SEQ ID NO:

11.

3. The variant of claim 1, wherein the variant has an amino acid substitution at a position corresponding to R282 of SEQ ID NO:

11.

4. The variant of claim 3, wherein the position corresponding to R282 of SEQ ID NO:

11 is substituted with any amino acid except for F, Y, I, and V.

5. The variant of claim 3, wherein the position corresponding to R282 of SEQ ID NO:

11 is substituted with K, T, Q, L, S, M, C, N, G or A.

7. A DNA encoding a variant enzyme according to claim 1.

8. A vector comprising the DNA according to claim 7.

9. A cell transformed with the vector according to claim 8.

10. A method, comprising:

reacting a variant enzyme of claim 1 with a DNA comprising one or more nucleotides selected from the group consisting of 5-β

glucosylhydroxymethylcytosine (5β

ghmC) and hydroxymethylcytosine, for fragmenting the DNA.

11. The method of claim 10, further comprising:

determining at least one of the location of and the amount of 5 hmC or 5β

ghmC in the DNA.

12. The method of claim 10, wherein said method comprise:

reacting the DNA with β

glucosyltransferase (β

GT) prior to reacting the variant enzyme with the DNA, thereby converting any hydroxymethylcytosines in the DNA to 5-β

glucosylhydroxymethylcytosines.

13. The method of claim 10, further comprising:

sequencing the DNA to create a hydroxymethylome map of the DNA.

14. The method of claim 10, wherein the DNA is part or all of a genome.

15. The method of claim 10, wherein the method comprises determining the presence or absence of 5 hmC or 5β

ghmC at a predetermined position in the DNA.

6. The variant of clam 3, wherein the position corresponding to R282 of SEQ ID NO:

11 is substituted with a G or A.

16. A method, comprising:

(a) obtaining a library of non-naturally occurring variants of a wild type restriction enzyme wherein the wild type restriction enzyme is defined by SEQ ID NO;

20, and wherein the variant has at least 90% sequence identity with SEQ ID NO;

20;

(b) assaying for cleavage specificity of the variant enzymes for 5β

GhmC and for 5 mC; and

(c) selecting a variant having at least 2 fold increase in selectivity for 5β

ghmC versus 5 mC compared to the wild type restriction enzyme.

17. The method of claim 16, wherein the variants have one or more amino acid substitutions within the amino acid sequence corresponding to SEQ ID NO:

20 or SEQ ID NO;

21.

18. The method of claim 16, wherein the variants have one or more amino acid substitutions outside the amino acid sequence corresponding to SEQ ID NO:

20 or SEQ ID NO;

21.