Programmable protein-DNA crosslinking for the direct capture and quantification of 5-formylcytosine
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Date
2019-06-04
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Abstract
5-Formylcytosine (5fC) is an epigenetic nucleobase
of mammalian genomes that occurs as intermediate
of active DNA demethylation. 5fC uniquely interacts
and reacts with key nuclear proteins, indicating functions
in genome regulation. Transcription-activator-like effectors
(TALEs) are repeat-based DNA binding proteins that
can serve as probes for the direct, programmable recognition
and analysis of epigenetic nucleobases. However, no
TALE repeats for the selective recognition of 5fC are available,
and the typically low genomic levels of 5fC represent
a particular sensitivity challenge. We here advance TALEbased
nucleobase targeting from recognition to covalent
crosslinking. We report TALE repeats bearing the ketoneamino
acid p-acetylphenylalanine (pAcF) that universally
bind all mammalian cytosine nucleobases, but selectively
form diaminooxy-linker-mediated dioxime crosslinks to
5fC. We identify repeat-linker combinations enabling single
CpG resolution, and demonstrate the direct quantification
of 5fC levels in a human genome background by covalent
enrichment. This strategy provides a new avenue to
expand the application scope of programmable probes
with selectivity beyond A, G, T and C for epigenetic studies.