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Technology

MDB proteins recognize symmetrically methylated CpG dinucleotides in double stranded DNA and if paired with sequence-specific probe DNA can detect methylation with high-resolution without chemical conversion or DNA sequencing. A high affinity protein may cover significantly more DNA sites leading to sequence specificity. A human methyl binding domain 2 (hMBD2) polypeptide library was created using error-prone PCR and was subsequently screened for high affinity hMBD2 polypeptides. This technology used a yeast species, Saccharomyces cerevisiae, to characterize binding properties of the hMBD2 polypeptides by displaying the proteins on the cell surface then measuring binding with fluorescence. The binding affinity of a hMBD2 polypeptide to a single methylated CpG site was doubled when compared to wild-type with just five amino acid substitutions.  This affinity can be further improved to a six-fold higher affinity when these modified polypeptides are concatenated three times as a GFP fusion.  This concatenated modified hMBD2 polypeptide is the highest affinity reagent for DNA methylation detection ever reported.