MS

thesisMethylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring Methyl Binding Domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG), and subsequently recruit other chromatin-modifying proteins to suppress transcription. This intrinsic property of MBD protein has been used to differentiate mCpG from nonmethylated CpG in conventional pulldown assays. However, the pulldown assay by itself does not provide information regarding the fraction of methylated DNA in the sample, the affinity of the MBD protein to different methylation patterns, and the relative positions of the mCpG sites. We focus on proof-of-concept studies to show that using real-time assay methods, recombinant proteins MBD2-venus and 1xMBD recognize hemimethylation and symmetrical methylation of CpG sequence, while displaying greater affinity for the symmetrical methylation motif. Real-time assay of protein binding was performed to characterize the kinetics of MBD2-Venus, 1xMBD and 1xMBD-GFP proteins. Two assay methods, SPR and fluorescence microarray, were used to evaluate the sensitivity and selectivity of MBD proteins binding to methylated dsDNA. The best-behaved MBD protein was chosen for subsequent fluorescence micrroarray studies. Recombinant 1xMBD-GFP was tested after fusion of the GFP gene downstream of the 1xMBD domain, but binding response and dissociation constant (KD) values suggest that the affinity of 1xMBD protein for mCpG was altered as a result of GFP insertion. MBD2-Venus was used for the fluorescence microarray experiment to interrogate the methylation status of Cy5-labeled dsDNA. In SPR studies of in situ hybridized dsDNA, for single symmetrical mCpG the KD values for 1xMBD-GFP and MBD2-Venus were found to be 1.68|iM and 76nM, respectively, while the KD of MBD2-Venus in fluorescence microarray assay was 0.7±0.1jaM. The difference in KD values is primarily due to the different surface chemistry between the SPR sensor and the microarray slide for fluorescence detection. However, the fluorescence microarray yielded sufficient sensitivity and selectivity to both single and double symmetrical methylation motifs of the CpG dinucleotide. Based on the current results, future work should expand to assays of larger DNA from tissue samples, as well as improving the fluorescence microarray design to reduce background noise and accommodate more capture probe arrays