CONVERSION OF ALPHA-HYDROXYALKYLATED RESIDUES IN BIOMOLECULES USING METHYLTRANSFERASES

• US 20120088238A1
• Filed: 04/01/2010
• Published: 04/12/2012
• Est. Priority Date: 04/02/2009
• Status: Active Grant

• Current Assignee: Vilnius University
• Original Assignee: Biotechnologijos Institutas
• Inventors: Klimasauskas, Saulius, Liutkeviciute, Zita, Kriukiene, Edita
• Granted Patent: US 8,889,352 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: 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
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2333/91011 Methyltransferases (general) (2.1.1.)

Abstract

The present invention relates to targeted conversion of alpha-hydroxyalkylated residues in biomolecules in the presence of a directing methyltransferase, namely to targeted removal of the alpha-hydroxyalkyl moieties to give unmodified residues, or targeted derivatization of the alpha-hydroxyalkyl groups by covalent coupling of non-cofactor compounds represented by formula HQ-LX1 wherein X represents a functional group or a reporter group attached via a linker moiety L, and QH is selected from HS—, HSe—, HO—H_2N—, HN₃ or HCN in the presence of a directing methyltransferase. Further development of the method of targeted conversion comprises methods for targeted labeling a biomolecule and method for detecting hydroxymethylated target sites in a biomolecule according to the present invention.

Claims

1. A method of using a cofactor-free methyltransferase for targeted conversion of a modified biomolecule, the method comprising

removing at a methyltransferase target site a modifying moiety of formula —

CH(OH)—

R, wherein R is hydrogen or C₁-C₁₂-alkyl, from the modified biomolecule using a cofactor-free methyltransferase resulting in targeted conversion of the modified biomolecule into an unmodified biomolecule.

3. The method of claim 1 or claim 2 wherein said biomolecule is a nucleic acid molecule, and said methyltransferase is a DNA cytosine-5 methyltransferase selected from the group consisting of M.HhaI, M.SssI, and M.HpaII or derivatives thereof.

15. The method of claim 1 wherein R in the modifying moiety being removed is hydrogen.

16. The method of claim 1 wherein R in the modifying moiety being removed is a lower alkyl.

17. The method of claim 1 wherein R in the modifying moiety being removed is —

CH₃.

21. The method of claim 3 wherein the nucleic acid molecule is DNA.

2. A method for targeted conversion of a modified biomolecule, comprising

incubating the modified biomolecule bearing a modifying moiety of formula —

CH(OH)—

R, wherein R is hydrogen or C₁-C₁₂-alkyl, with a cofactor-free directing methyltransferase under conditions compatible with enzymatic activity of the methyltransferase, and

obtaining targeted conversion resulting from;

i) covalent removal of said modifying moiety at the target site;

or

ii) derivatization of said modifying moiety at the target site by covalent coupling of non-cofactor nucleophilic compound(s) of general formula HQ-LX, wherein X represents a functional group or a reporter group attached via a linker L, and Q is selected from S, Se, O, N, or C.

4. The method of claim 2, wherein the modified biomolecule is a naturally or artificially modified biomolecule, and wherein R is hydrogen or —

CH₃ and Q is S or Se.

5. The method of claim 2 further comprising directly or subsequently incorporating a reporter group suitable as a label and allowing identification of the labeled molecule among other unlabeled molecules.

6. The method of claim 5, wherein the label is selected from fluorophores, fluorescence quenchers, chromophores, affinity tags, stable paramagnetic groups, groups containing radioactive or stable rare isotopes, groups containing heavy atoms suitable for phasing X-ray diffraction data, crosslinking agents, nucleic acids cleaving groups, haptens, nanoparticles, beads and combinations thereof.

11. The method of claim 5, wherein the coupled compound or a label in a derivatized biomolecule is identified by DNA sequencing, hybridization, mass spectrometry or analysis of nucleoside composition by enzymatic fragmentation and chromatography.

18. The method of claim 2 wherein R in the modifying moiety is hydrogen.

19. The method of claim 2 wherein R in the modifying moiety is a lower alkyl.

20. The method of claim 2 wherein R in the modifying moiety is —

CH₃.

7. A method for detecting hydroxymethylated target sites in a biomolecule, comprising

derivatizing or labeling the biomolecule by coupling non-cofactor nucleophilic compounds of general formula HQ-LX, wherein X represents a functional group or a reporter group attached via a linker L, and Q is selected from S, Se, O, N, or C in the presence of a cofactor-free methyltransferase, and

detecting whether the target sites of said methyltransferase have been modified, wherein modification of the target site of said methyltransferase is indicative of the presence of hydroxymethylated target site.

8. The method of claim 7, wherein the coupled compound interferes with nucleic acid amplification at the recognition sites of the methyltransferase;

and hydroxymethylated target sites are detected by testing whether amplification of the nucleic acid molecule at the recognition sites of the methyltransferase has been retarded.

9. The method of claim 7, wherein the coupled compound comprises a fluorescent label;

and hydroxymethylated target sites are detected by measuring the presence or amount of fluorescence in said nucleic acid molecule.

10. The method of claim 7, wherein the coupled compound is added to a 5-hydroxymethylcytosine residue in DNA and cannot be added to a 5-methylcytosine or a cytosine residue.

22. The method of claim 7 wherein the coupled compound or a label in a derivatized biomolecule is identified by DNA sequencing, hybridization, mass spectrometry or analysis of nucleoside composition by enzymatic fragmentation and chromatography.

23. The method of claim 5 or claim 22 wherein the derivatized biomolecule is DNA.

12. A kit comprising a cofactor-free directing methyltransferase or a cofactor-free directing methyltransferase and non-cofactor nucleophilic compound(s) in separate containers, and

instructions for use to perform targeted conversion of a modified biomolecule or to detect hydroxymethylated target sites in a biomolecule.

13. The kit of claim 12, further comprising scavenging compound(s) in separate containers.

14. A 2′

-deoxycytidine derivative formed in DNA, which is 5-(Se-selenocysteinyl)methyl-2′

-deoxycytidine.