(541d) DNA Methylation Detection with an Engineered Protein That Binds Hemi-Methylated DNA

DNA methylation, specifically the methylation of cytosine bases, is an important biomarker, as abnormal DNA methylation patterns are found in many different types of cancer. Currently, a small number of cancer hospitals evaluate the methylation status of the MGMT gene promoter to determine the best course of treatment for patients with glioblastoma. However, improved methylation detection techniques are required in order to expand the availability of such testing to more patients. Methyl-CpG-binding domain (MBD) proteins bind specifically to methylated DNA sequences, and many assays have been developed that use these proteins in methylation profiling of DNA. The wild-type proteins in the MBD family bind specifically to symmetrically methylated CpG dinucleotides. Here, we describe the engineering of a new MBD that binds to hemi-methylated DNA but not unmethylated DNA, which allows for the detection of a methylated target sequence hybridized to an unmethylated DNA probe. The binding affinity of this engineered protein toward a DNA sequence with a single hemi-methylated CpG dinucleotide was evaluated, and a dissociation constant of 5.6 ± 1.4 nM was achieved. The protein was expressed in soluble form as a fusion protein with green fluorescent protein, and performance in distinguishing between hemi-methylated (methylated target DNA bound to an unmethylated probe) and unmethylated (unmethylated target DNA bound to an unmethylated probe) DNA in an interfacial binding assay was demonstrated. Using this protein, an assay was developed and optimized to determine the methylation status of the MGMT gene promoter by hybridizing the target DNA to a probe on a surface and determining the methylation status of the captured target based on the amount of MBD bound. Such an assay is useful in guiding treatment plans for glioblastoma patients because MGMT promoter methylation is correlated with better response to alkylating chemotherapeutics such as temozolomide.