Design and Application of DNA Modification-Specific Transcription-Activator-Like Effectors.

Transcription-activator like effectors (TALEs) are DNA-binding proteins used for genome targeting. TALEs contain a central domain of concatenated repeats, of which each selectively recognizes one nucleobase at the DNA major groove. Based on this simple and predictable interaction with little context dependence, TALEs offer programmable targeting of user-defined DNA sequences. Since many epigenetic DNA modifications protrude into the DNA major groove, natural and engineered TALE repeats can provide "epigenetic" selectivity, making TALEs a flexible platform to design probes for the analysis of epigenetic DNA modifications. Here, we describe guidelines for the design of TALE proteins with selectivity for epigenetic cytosine 5-modifications, the validation of their interaction with modified DNA nucleobases, and their employment in affinity enrichment assays. These techniques enable quantification of epigenetic nucleobases in user-defined genomic DNA sequences with nucleotide and strand resolution.

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

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 TALE-based nucleobase targeting from recognition to covalent cross-linking. We report TALE repeats bearing the ketone-amino acid p-acetylphenylalanine (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.

Complete, Programmable Decoding of Oxidized 5-Methylcytosine Nucleobases in DNA by Chemoselective Blockage of Universal Transcription-Activator-Like Effector Repeats.

5-Methylcytosine (5mC) and its oxidized derivatives are regulatory elements of mammalian genomes involved in development and disease. These nucleobases do not selectively modulate Watson-Crick pairing, preventing their programmable targeting and analysis by traditional hybridization probes. Transcription-activator-like effectors (TALEs) can be engineered for use as programmable probes with epigenetic nucleobase selectivity. However, only partial selectivities for oxidized 5mC have been achieved so far, preventing unambiguous target binding. We overcome this limitation by destroying and re-inducing nucleobase selectivity in TALEs via protein engineering and chemoselective nucleobase blocking. We engineer cavities in TALE repeats and identify a cavity that accommodates all eight human DNA nucleobases. We then introduce substituents with varying size, flexibility, and branching degree at each oxidized 5mC. Depending on the nucleobase, substituents with distinct properties effectively block TALE-binding and induce full nucleobase selectivity in the universal repeat. Successful transfer to affinity enrichment in a human genome background indicates that this approach enables the fully selective detection of each oxidized 5mC in complex DNA by programmable probes.

The N6-Position of Adenine Is a Blind Spot for TAL-Effectors That Enables Effective Binding of Methylated and Fluorophore-Labeled DNA.

Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins widely used for genome targeting. TALEs consist of multiple concatenated repeats, each selectively recognizing one nucleobase via a defined repeat variable diresidue (RVD). Effective use of TALEs requires knowledge about their binding ability to epigenetic and other modified nucleobases occurring in target DNA. However, aside from epigenetic cytosine-5 modifications, the binding ability of TALEs to modified DNA is unknown. We here study the binding of TALEs to the epigenetic nucleobase N6-methyladenine (6mA) found in prokaryotic and recently also eukaryotic genomes. We find that the natural, adenine (A)-binding RVD NI is insensitive to 6mA. Model-assisted structure-function studies reveal accommodation of 6mA by RVDs with altered hydrophobic surfaces and abilities of hydrogen bonding to the N6-amino group or N7 atom of A. Surprisingly, this tolerance of N6 substitution was transferrable to bulky N6-alkynyl substituents usable for click chemistry and even to a large rhodamine dye, establishing the N6 position of A as the first site of DNA that offers label introduction within TALE target sites without interference. These findings will guide future in vivo studies with TALEs and expand their applicability as DNA capture probes for analytical applications in vitro.

Interrogating Key Positions of Size-Reduced TALE Repeats Reveals a Programmable Sensor of 5-Carboxylcytosine.

Transcription-activator-like effector (TALE) proteins consist of concatenated repeats that recognize consecutive canonical nucleobases of DNA via the major groove in a programmable fashion. Since this groove displays unique chemical information for the four human epigenetic cytosine nucleobases, TALE repeats with epigenetic selectivity can be engineered, with potential to establish receptors for the programmable decoding of all human nucleobases. TALE repeats recognize nucleobases via key amino acids in a structurally conserved loop whose backbone is positioned very close to the cytosine 5-carbon. This complicates the engineering of selectivities for large 5-substituents. To interrogate a more promising structural space, we engineered size-reduced repeat loops, performed saturation mutagenesis of key positions, and screened a total of 200 repeat-nucleobase interactions for new selectivities. This provided insight into the structural requirements of TALE repeats for affinity and selectivity, revealed repeats with improved or relaxed selectivity, and resulted in the first selective sensor of 5-carboxylcytosine.