Programmable Protein–DNA Cross-Linking for the Direct Capture and Quantification of 5-Formylcytosine

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-activatorlike 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 TALE-based nucleobase targeting from recognition to covalent cross-linking. We report TALE repeats bearing the ketone-amino acid pacetylphenylalanine (pAcF) that universally bind all mammalian cytosine nucleobases, but selectively form diaminooxy-linker-mediated dioxime cross-links 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.

Advances and Opportunities in Epigenetic Chemical Biology

“…While there is not yet a specific TALE available for 5fC, the Summerer group has developed an alternate strategy. Using a TALE containing a p ‐acetylphenylalanine noncanonical amino acid (ncAA) incorporated by genetic code expansion (discussed in detail below), the formyl moiety of the 5fC will cross‐link to the ncAA, enabling 5fC detection [39] . The 5fC modification can also be detected by specific chemical modification to the formyl moiety [40] .…”

“…Using a TALE containing a p-acetylphenylalanine noncanonical amino acid (ncAA) incorporated by genetic code expansion (discussed in detail below), the formyl moiety of the 5fC will cross-link to the ncAA, enabling 5fC detection. [39] The 5fC modification can also be detected by specific chemical modification to the formyl moiety. [40] Further, TALEs have also been used in nontraditional fashions.…”

Facile Clamp-Assisted Ligation Strategy for Direct Discrimination and Background-Free Quantification of Site-Specific 5-Formylcytosine

“…Nevertheless, such strategies require harsh bisulfite treatment, where the acidic and thermal conditions may result in some DNA degradation. − Quite recently, the Yi group has reported the elegant bisulfite-free strategies by specifically label 5fC with some functional compounds, , which could map 5fC under mild conditions. However, as for sensitively detecting known 5fC sites of biomedical interest, deep sequencing is costly and time-consuming, prohibiting its routine clinical use. , Although great efforts toward simple and accurate detection of site-specific 5fC have been made, − there still remains two roadblocks on the way toward this goal. On one hand, 5fC is of very low abundance in genomic DNA, which requires ultrahigh sensitivity of the detection method.…”