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.
Genexpression ist stark durch spezifische Muster von genomischem 5-Methylcytosin (mC) reguliert, aber die direkte Detektion dieser Modifizierung an benutzerdefinierten Genorten ist nur eingeschränkt mçglich, unter anderem, weil es keine Proteine gibt, die zwischen mC und Cytosin (C) unterscheiden kçnnen und zugleich eine inhärente, programmierbare Sequenzselektivität bieten. Die hier vorgestellte Methode zur mC-Detektion beruht auf mC-abhängiger Steuerung der DNA-Replikation und zeigt eine unerwartet ausgeprägte mC-Sensitivität von Transkriptionsaktivator-artigen Effektoren (TALEs). Damit kann der Methylierungsstatus und -grad an einzelnen C-Positionen von Oligonucleotiden für die N-terminalen und zentralen Regionen von TALEs eindeutig evaluiert werden, unabhängig von der Zielsequenz. Dies spricht für eine breite Anwendbarkeit und Robustheit der Methode zur hochaufgelçsten mC-Detektion und ermçglicht die Erkennung von mC an einzelnen Positionen in großen eukaryotischen Genomen.
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