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.

Anti-Markovnikov alkene oxidation by metal-oxo-mediated enzyme catalysis.

Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidation of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration.

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.

Isolation of Human Genomic DNA Sequences with Expanded Nucleobase Selectivity.

We report the direct isolation of user-defined DNA sequences from the human genome with programmable selectivity for both canonical and epigenetic nucleobases. This is enabled by the use of engineered transcription-activator-like effectors (TALEs) as DNA major groove-binding probes in affinity enrichment. The approach provides the direct quantification of 5-methylcytosine (5mC) levels at single genomic nucleotide positions in a strand-specific manner.

TALEored Epigenetics: A DNA-Binding Scaffold for Programmable Epigenome Editing and Analysis.

Epigenetic modification of the cytosine 5-position is an important regulator of gene expression with essential roles in genome stability, development, and disease. In addition to 5-methylcytosine (mC), the oxidized mC derivatives 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine (hmC, fC, and caC) have recently been discovered. These are intermediates of an active demethylation pathway but might also represent new epigenetic marks with individual biological roles.

Deciphering Epigenetic Cytosine Modifications by Direct Molecular Recognition.

Epigenetic modification at the 5-position of cytosine is a key regulatory element of mammalian gene expression with important roles in genome stability, development, and disease. The repertoire of cytosine modifications has long been confined to only 5-methylcytosine (mC) but has recently been expanded by the discovery of 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine. These are key intermediates of active mC demethylation but may additionally represent new epigenetic marks with distinct biological roles.

Programmable sensors of 5-hydroxymethylcytosine.

5-Hydroxymethylcytosine (hmC), the sixth base of the mammalian genome, is increasingly recognized as an epigenetic mark with important biological functions. We report engineered, programmable transcription-activator-like effectors (TALEs) as the first DNA-binding receptor molecules that provide direct, individual selectivities for cytosine (C), 5-methylcytosine (mC), and hmC at user-defined DNA sequences. Given the wide applicability of TALEs for programmable targeting of DNA sequences in vitro and in vivo, this provides broad perspectives for epigenetic research.

Achieving single-nucleotide resolution of 5-methylcytosine detection with TALEs.

We report engineered transcription-activator-like effectors (TALEs) as the first DNA-binding molecules that detect 5-methylcytosine (mC) at single-nucleotide resolution with fully programmable sequence selectivity. This is achieved by a design strategy such that a single cytosine (C) in a DNA sequence is selectively interrogated for its mC-modification level by targeting with a discriminatory TALE repeat; other Cs are ignored by targeting with universal-binding TALE repeats.

Programmable and highly resolved in vitro detection of 5-methylcytosine by TALEs.

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.