This invention improves DNA methylation detection and may be used for the diagnosis, prognosis, or better informed treatment of a number of diseases including, but not limited to, gliomas, colon cancer, and prostate cancer.
Methylation is a type of epigenetic modification involved in DNA condensation and gene inactivation and divergent methylation patterns contribute to the pathogenesis of disease including cancers. Promoter methylation has great predictive, prognostic and diagnostic value. For example, therapeutic response of breast cancer patients may be determined as patients with methylation dependent silencing of the BRCA1 gene have tumors sensitive to cisplatin. Therefore, accurate methods for DNA methylation detection are desirable. Current methods include bisulfite conversion which degrades over 90% of the DNA sample leading to inaccurate results and immunoprecipitation methods which require large samples and offer low resolution. Recently, methods using methyl-binding domain (MBD) proteins have been developed but are not sequence specific. In this invention high affinity MBD protein variants were determined for the purpose of increasing sequence specificity.
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.
- No chemical conversion or DNA sequencing needed
- Increased affinity means high resolution