Means and Methods for Detecting Methylated DNA

Abstract

The present application relates to a nucleic acid molecule having a nucleotide sequence encoding a bifunctional polypeptide comprising the DNA-binding domain of a protein belonging to the family of Methyl-CpG binding proteins (MBDs) and the Fc portion of an antibody. In addition, vectors and host cells which comprise said nucleic acid molecule and polypeptides which are encoded by said nucleic acid molecule as well as processes for producing said polypeptide are disclosed. Moreover, the present application provides an antibody specifically binding said polypeptide and compositions, in particular diagnostic compositions comprising the nucleic acid molecule(s), vector(s), host cell(s), polypeptide(s) or antibodie(s) of the present application. Furthermore, methods and uses employing the polypeptides of the present invention for detecting methylated DNA, in particular in tumorous tissue or tumor cells are provided.

1 . A reagent for binding methylated DNA comprising:
 a first polypeptide and a second polypeptide each comprising: (i) a methyl-DNA-binding domain of an MBD2 protein; (ii) an Fc portion of an antibody and (iii) a flexible peptide linker, wherein the first polypeptide and second polypeptide each have the methyl-DNA-binding domain of the MBD2 protein fused to the Fc portion of an antibody through the flexible peptide linker and the Fc portion of an antibody of the first polypeptide is bonded to the Fc portion of an antibody of the second polypeptide; and   a bivalent binding site for methylated DNA comprising the methyl-DNA-binding domain of the first polypeptide in proximity to the methyl-DNA-binding domain of the second polypeptide, wherein the bivalent binding site can bind two methylated CpG residues that are on the same strand of a double stranded DNA molecule or on different strands of a double stranded DNA molecule.   
     
     
         2 . The reagent of  claim 1 , wherein the capacity to bind to methylated DNA is dependent on the degree of methylation. 
     
     
         3 . The reagent of  claim 1 , wherein the capacity to bind to methylated DNA is dependent on salt concentration. 
     
     
         4 . The reagent of  claim 1 , wherein the MBD2 protein is a human MBD2 protein. 
     
     
         5 . The reagent of  claim 1 , wherein the reagent can detect methylated DNA in a sample of less than 10 ng of genomic DNA. 
     
     
         6 . The reagent of  claim 1 , wherein the reagent can detect methylated DNA in a sample of less than 5 ng of genomic DNA. 
     
     
         7 . The reagent of  claim 1 , wherein the reagent can detect methylated DNA in a sample of less than 1 ng of genomic DNA. 
     
     
         8 . A composition comprising the reagent of  claim 1 .