Conversion of alpha-hydroxyalkylated residues in biomolecules using methyltransferases

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—H 2 N—, HN 3 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.

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 —CH 2 CH(CO 2 H)—, (poly)ethyleneglycol chains (CH 2 CH 2 O) 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 —CH 2 CH 2 OH, —CH 2 CH(CO 2 H)NH 2 , -5′-deoxyadenosine, —CH 2 CH 2 NH 2 , —CH 2 CH(OH)CH(OH)CH 2 SH, and —OH. 
     
     
         9 - 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 . (canceled)