Direct DNA methylation profiling using methyl binding domain proteins

Yinni Yu, Steve Blair, David Gillespie, Randy Jensen, David Myszka, Ahmed H. Badran, Indraneel Ghosh, Alexander Chagovetz

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

Methylation 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. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemimethylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the KD for 1xMBD-GFP ranged from 106 to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The KD values for nonsymmetrical methylation motifs were consistently greater (>2 μM), but the binding selectivity between symmetric and hemimethylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density.

Original languageEnglish (US)
Pages (from-to)5012-5019
Number of pages8
JournalAnalytical chemistry
Volume82
Issue number12
DOIs
StatePublished - Jun 15 2010

ASJC Scopus subject areas

  • Analytical Chemistry

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    Yu, Y., Blair, S., Gillespie, D., Jensen, R., Myszka, D., Badran, A. H., Ghosh, I., & Chagovetz, A. (2010). Direct DNA methylation profiling using methyl binding domain proteins. Analytical chemistry, 82(12), 5012-5019. https://doi.org/10.1021/ac1010316