CONVERSION OF ALPHA-HYDROXYALKYLATED RESIDUES IN BIOMOLECULES USING METHYLTRANSFERASES

• US 20170067093A1
• Filed: 10/13/2016
• Published: 03/09/2017
• Est. Priority Date: 04/02/2009
• Status: Abandoned Application

• Current Assignee: Vilnius University
• Original Assignee: Vilnius University
• Inventors: Klimasauskas, Saulius, Liutkeviciute, Zita, Kriukiene, Edita
• Application Number:
  US15/293,232
• Publication Number:
  US 20170067093A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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-LX, wherein X represents a functional group or a reporter group attached via a linker moiety L, and QH is selected from HS—, HSe—, HO—H₂N—, 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 composition comprising a cofactor-free methyltransferase and a non-cofactor nucleophilic compound, where the non-cofactor nucleophilic compound is general formula HQ-LX, where X represents a functional group and/or a reporter group attached by linker L to Q, and Q is selected from the group consisting of S, Se, O, N, and C.

2. The composition of claim 1 where the cofactor-free methyltransferase is a DNA cytosine-5 methyltransferase selected from the group consisting of M.HhaI, M.SssI, and M.HpaII or derivatives thereof.

3. The composition of claim 1 where linker L is selected from the group consisting of —

CH₂CH(CO₂H)—

, (poly)ethyleneglycol chains (CH₂CH₂O)_n where n=1-100, a covalent bond, and a linear, cyclic, and/or aromatic alkyl optionally comprising at least one of NHCO, O, or S.

4. The composition of claim 1 where X comprises at least one reactive group selected from the group consisting of a primary amino group, a thiol group, a 1,2-diol group, a haloacetamide group, a maleimide group, an aldehyde group, a ketone group, an azido group, an alkyne group, a 1,3-diene function, a dienophilic function, an arylhalide group, a terminal alkyne group, an arylboronic acid group, a terminal haloalkyne group, a terminal silylalkyne group and a protected amino, thiol, 1,2-diol, hydrazino, hydroxyamino, aldehyde, ketone, and 1,2-aminothiol group.

5. The composition of claim 1 where X comprises at least one reporter selected from the group consisting of a heavy atom or heavy atom cluster, a radioactive or stable rare isotope, a residue of a member selected from fluorophores, fluorescence quenchers, or chromophores, an affinity label, a spin label, a group containing a radioactive or stable rare isotope, a group containing a heavy atom, a crosslinking agent, a nucleic acid cleaving group, a hapten, a nanoparticle, a bead, and combinations thereof.

6. The composition of claim 1 where X comprises an affinity label and the composition further comprises a bead capable of binding the affinity label.

7. The composition of claim 1 further comprising a scavenging compound.

8. The composition of claim 1, where -LX is selected from the group consisting of —

CH₂CH₂OH, —

CH₂CH(CO₂H)NH₂, -5′

-deoxyadenosine, —

CH₂CH₂NH₂, —

CH₂CH(OH)CH(OH)CH₂SH, and —

OH.

9-30. -30. (canceled)

31. A method for detecting an endogenously hydroxymethylated residue in a biomolecule, the method comprising

derivatizing or labeling a hydroxymethylated group of the endogenously hydroxymethylated residue in the biomolecule to result in a derivatized or labeled biomolecule, and

detecting the derivatized or labeled biomolecule, where derivatization or labeling of the biomolecule is indicative of the presence of the hydroxymethylated residue in the biomolecule.

32. The method of claim 31 further comprising isolating the labeled biomolecule using the derivatized or labeled hydroxymethylated residue.

33. The method of claim 32 where the derivatizing or labeling the hydroxymethylated group of the endogenously hydroxymethylated residue results in addition of an affinity label, and where the affinity label is used for isolating the labeled biomolecule.

34. The method of claim 33 where the affinity label is biotin.

35. The method of claim 31 where the biomolecule is a nucleic acid molecule selected from the group consisting of DNA, RNA, and DNA/RNA hybrids.

36. The method of claim 35 where the DNA is genomic DNA.

37. The method of claim 31 where the hydroxymethylated residue is 5-hydroxymethylcytosine or 5-hydroxymethyluracil.

38. The method of claim 37 where the method distinguishes hydroxymethylated, methylated, and non-methylated residues.

39. The method of claim 31 where the hydroxymethylated group of the hydroxymethylated residue is derivatized or labeled by coupling of a non-cofactor nucleophilic compound of general formula HQ-LX, where X represents a functional group or a reporter group attached by linker L, and Q is selected from the group consisting of S, Se, O, N, and C in the presence of a cofactor-free methyltransferase.

40. The method of claim 39 where the reporter group is a fluorescent label, and the hydroxymethylated residue is detected by measuring the presence or amount of fluorescence in the biomolecule.

41. The method of claim 39 where X comprises heavy atoms or heavy atom clusters, radioactive or stable rare isotopes, residue of a fluorophore, fluorescence quencher, chromophore, affinity tag, spin label, crosslinking agent, nucleic acid cleaving groups, haptens, nanoparticles, and/or beads.

42. The method of claim 31 where detecting the derivatized or labeled hydroxymethylated residue in the labeled biomolecule comprises a process selected from the group consisting of DNA sequencing, DNA hybridization, mass spectrometry, analysis of nucleoside composition by enzymatic fragmentation and chromatography, and combinations thereof.

43. The method of claim 31 where the labeled biomolecule is detected by an antibody specifically binding to the label of the labeled biomolecule or by avidin or streptavidin specifically binding to the label of the labeled biomolecule.

44. A kit comprising

a cofactor-free directing methyltransferase or a cofactor-free directing methyltransferase and non-cofactor nucleophilic compound(s) in separate containers,

an affinity label,

a bead capable of binding the affinity label, and

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

45. The kit of claim 44 further comprising scavenging compound(s) in separate containers.

46-55. -55. (canceled)