title: Programmable and highly resolved in vitro detection of 5-methylcytosine by TALEs.
abstract: Gene expression is extensively regulated by specific patterns of genomic 5-methylcytosine (mC), but the ability to directly detect this modification at user-defined genomic loci is limited. One reason is the lack of molecules that discriminate between mC and cytosine (C) and at the same time provide inherent, programmable sequence-selectivity. Programmable transcription-activator-like effectors (TALEs) have been observed to exhibit mC-sensitivity in vivo, but to only a limited extent in vitro. We report an mC-detection assay based on TALE control of DNA replication that displays unexpectedly strong mC-discrimination ability in vitro. The status and level of mC modification at single positions in oligonucleotides can be determined unambiguously by this assay, independently of the overall target sequence. Moreover, discrimination is reliably observed for positions bound by N-terminal and central regions of TALEs. This indicates the wide scope and robustness of the approach for highly resolved mC detection and enabled the detection of a single mC in a large, eukaryotic genome.
authors.affiliation: Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz (Germany).
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Kubik
authors.forename: Moritz J
authors.initials: MJ
authors.lastname: Schmidt
authors.forename: Johanna E
authors.initials: JE
authors.lastname: Penner
authors.forename: Daniel
authors.initials: D
authors.lastname: Summerer
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques / Q000379:methods*
chemical_list: D044503:5-Methylcytosine; D004247:DNA
keywords: 5-methylcytosine; TAL effectors; epigenetics; programmable protein scaffolds; protein-DNA interactions