Direct Detection of Hypermethylation in Cancer

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DESCRIPTION (provided by applicant): The long-term objective of the proposed research project is to provide a robust, sensitive, and rapid method for the direct detection of CpG island methylation in the promoter region of specific genes implicated in cancer. Cytosine methylation occurs at CpG dinucleotides in 70-80 percent of the human genome, most often in repetitive genomic regions. On the other hand, CpG islands, defined as short sequences with statistically high CpG content, present in the promoter region of many genes (60 percent) are primarily protected from methylation in normal tissues. These CpG islands have been found to be methylated in cancer, leading to transcriptional repression. Recent experiments provide strong correlation between CpG hypermethylation at promoter sites of numerous genes and the incidence of cancer, thus making specific promoter hypermethylation a valuable marker for early detection. Current methods for detection of specific CpG island methylation rely on extensive bisulfite treatment of methylated DNA followed by PCR-based amplification, sequencing, or microarray techniques. These current methods, though powerful, are also laborious, time-intensive and expensive for characterizing known sites of hypermethylation. Towards the goal of rapidly determining promoter CpG hypermethylation, we will apply our newly developed technology called SEquence Enabled Reassembly (SEER) of proteins. The SEER system allows for the recognition of specific sequences of double-stranded DNA that result in the concomitant assembly of functional protein reporters (green fluorescent protein and beta-lactamase). In the proposed detection of specific CpG hypermethylation, we will target CpG islands utilizing the methyl-CpG binding domain (MBD) of the MBD2 protein, while targeting the correct promoter sequence utilizing a designed zinc-finger. Our approach has the potential to provide a sensitive turn-on sensor for directly reporting upon CpG methylation at known promoter sites. This approach, if successful, will rapidly distinguish between normal and cancerous tissues in a clinical setting without the requirement for bisulfite treatment, PCR amplification, and sequencing. We will provide proof of concept by 1) designing and optimizing turn-on biosensors for detecting specific methylation events in model DNA constructs; and 2) designing and testing biosensors that target promoter regions of genes (BRCA1, CDH1, p15, p16, MGMT, GSTp1) implicated in cancer.