Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application.

Wild-type methyl-CpG-binding domain (MBD) proteins specifically bind symmetrically methylated DNA sequences, and assays have been developed that use these proteins for profiling DNA methylation. Here, we use directed evolution in the yeast surface display format to identify a new protein variant that binds hemi-methylated CpG dinucleotides.

Epigenetic silencing of genes that are important for DNA repair, cell cycle control, apoptosis, and cellular interactions with the extracellular matrix has been causally linked to several subtypes of cancer. Translating this knowledge of the implications of promoter methylation to wide and routine use in clinical pathology laboratories has been more challenging than the case of genetic analyses because epigenetic modifications do not change the underlying sequence of the affected nucleic acid, rendering polymerase chain reaction analysis alone uninformative. Two epigenotyping assays that detect promoter methylation are currently standard of care in treatment of two distinct tumor types in only a few top hospitals across the United States. Both rely on a harsh chemical step that degrades over 90% of tumor DNA samples, which are often available in limited quantities, and imparts the potential for false-negative or false-positive results if the reaction conditions are not exactly correct. Using nanotechnology and biotechnology to devise practical new analysis techniques that avoid the drawbacks of current techniques represents a powerful approach that is likely to significantly increase the clinical use of this class of biomarkers in the coming years. WIREs Nanomed Nanobiotechnol 2017, 9:e1407. doi: 10.1002/wnan.1407 For further resources related to this article, please visit the WIREs website.

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