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Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5‑Hydroxymethylcytosine/5-Methylcytosine CpG Dyads

5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve met...

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Published in:Journal of the American Chemical Society 2022-02, Vol.144 (7), p.2987-2993
Main Authors: Buchmuller, Benjamin C, Dröden, Jessica, Singh, Himanshu, Palei, Shubhendu, Drescher, Malte, Linser, Rasmus, Summerer, Daniel
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cited_by cdi_FETCH-LOGICAL-a417t-786c140233b8c199d287498dc4d75430876ea8789d5751eeca8bccc0bbf45e853
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container_issue 7
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container_title Journal of the American Chemical Society
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creator Buchmuller, Benjamin C
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description 5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.
doi_str_mv 10.1021/jacs.1c10678
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source American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list)
subjects 5-Methylcytosine - analogs & derivatives
5-Methylcytosine - chemistry
Directed Molecular Evolution
DNA - chemistry
DNA - isolation & purification
DNA Methylation
HEK293 Cells
Humans
Methyl-CpG-Binding Protein 2 - chemistry
Methyl-CpG-Binding Protein 2 - genetics
Peptide Fragments - chemistry
Peptide Fragments - genetics
Protein Domains
title Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5‑Hydroxymethylcytosine/5-Methylcytosine CpG Dyads
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