High-Affinity, Engineered Methyl Binding Domain (MBD) Proteins

Technology #17707

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Detection and quantification of methylated DNA binding to yeast displayed MBD proteins. (a) Yeast displaying MBD proteins were incubated with biotinylated, methylated DNA and a primary anti-c-Myc antibody followed by labeling with streptavidin, Alexa Fluor® 647 and an Alexa Fluor® 488 secondary antibody, respectively. (b) Flow cytometry dot plot showing 50 nM omo DNA and (c) 50 nM ooo DNA binding to wild-type hMBD2. (d) Equilibrium binding titration curves for determining the affinity of wild-type hMBD2 binding to DNA with various DNA methylation patterns. The mean fluorescence of the displaying yeast population is normalized and plotted versus DNA concentration. Fitting the data yields the equilibrium dissociation constant (Kd) for each oligo. Each reported value (Table I) is the average of three such biological replicates (only one shown). (e) Titration curves for wild-type MBD2, variant 1/4 and variant 2/5 binding to omo DNA. Leftward shift of the binding curve indicates higher affinity binding.Structural analysis of amino acid substitutions in MBD variant 2/5. (a) The addition of the para substituted hydroxyl group of tyrosine relative to the wild-type phenylalanine forms a new hydrogen bond to the DNA phosphodiester backbone. (b) Mutating lysine 161 to arginine introduces a guanidinium group capable of forming an additional hydrogen bond to the main chain carbonyl of aspartic acid 151 in addition to that the wild-type lysine makes to the main chain carbonyl of glycine 211. (c) The side chains of isoleucines 165 and 175 form a hydrophobic interaction at the end of β2 and beginning of β3. (d) Isoleucine 187 and leucine 193 share a hydrophobic interaction between β4 and α1 in the vicinity of three residues lysine 186 (backbone), arginine 188 (bases) and serine 189 (backbone) known to interact with the bound DNA strand.
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Inventors
Professor Hadley Johnson
Department of Chemical Engineering, MIT
External Link (hsikeslab.mit.edu)
Brandon Heimer
Department of Chemical Engineering, MIT
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Characterization and Directed Evolution of a Methyl Binding Domain Protein for High-Sensitivity DNA Methylation Analysis

PCT Patent Application WO 2016-210124

Characterization and Directed Evolution of a Methyl Binding Domain Protein for High-Sensitivity DNA Methylation Analysis

US Patent Pending
Publications
Characterization and directed evolution of a methyl-binding domain protein for high-sensitivity DNA methylation analysis
Protein Engineering, Design & Selection, September 18, 2015

Applications

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.  

Problem Addressed

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.

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. 

Advantages

  • No chemical conversion or DNA sequencing needed
  • Increased affinity means high resolution