Semisynthetic cyclic peptides containing both non-proteinogenic building blocks, as the synthetic part, and a genetically encoded sequence amenable to DNA-based randomization hold great potential to expand the chemical space in the quest for novel bioactive peptides. Key to an efficient selection of novel binders to biomacromolecules is a robust method to link their genotype and phenotype. A novel bacterial cell surface display technology has been developed to present cyclic peptides composed of synthetic and genetically encoded fragments in their backbones. The fragments were combined by protein trans-splicing and intramolecular oxime ligation. To this end, a split intein half and an unnatural amino acid were displayed with the genetically encoded part on the surface of Escherichia coli. Addition of the synthetic fragment equipped with the split intein partner and an aminooxy moiety, as well as the application of a pH-shift protocol, resulted in the onsurface formation of the semisynthetic cyclic peptide. This approach will serve for the generation of cyclic peptide libraries suitable for selection by fluorescence-activated cell sorting, and more generally enables chemical modification of proteins on the bacterial surface.

Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dynamically reshapes the epigenome and transcriptome by depositing oxidized 5mC derivatives and initiating active DNA demethylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with temporal control in cells. We report activation of TETs in mammalian cells by incorporation of genetically encoded 4,5-dimethoxy-2-nitrobenzyl-L-serine as a transient active-site block, and its subsequent deprotection with light. Our approach enables precise insights into the impact of cancer-associated TET2 mutations on the kinetics of TET2 catalysis in vivo, and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA demethylation to chromatin and transcription regulation.

5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.

Cyclic peptides can be highly valuable as bioactive molecules, both for biomedical applications and in basic research. We introduce a new fragment-based approach to access cyclic peptide structures in which one fragment is of synthetic origin and the other is genetically encoded. The synthetic peptide, which can contain one or more non-proteinogenic building blocks, is coupled to the recombinantly expressed peptide through two bonds, one formed by protein trans-splicing with a split intein and the other by oxime ligation. Semisynthetic macrocycles were obtained with high efficiency for various sequences and ring sizes; they can be prepared in quantities sufficient for initial bioactivity tests. We also prepared lipidated and d-amino-acid-containing peptides that were inspired by the peptide antibiotic daptomycin. Such structures are not accessible by other methods that harness the power of simple genetic diversification in the DNA-encoded part of the peptide.

, the central epigenetic mark of mammalian DNA, playsf undamental roles in chromatin regulation. 5mC is written onto genomes by DNAm ethyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis.H owever,s tudying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo.W ereport light-control of DNMT catalysis by genetically encoding ap hotocaged cysteine as ac atalytic residue.T his enables translation of inactive DNMTs,their rapid activation by light-decaging,and subsequent monitoring of de novo DNAm ethylation. We providei nsights into how cancer-related DNMT mutations alter de novo methylation in vivo,a nd demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to ap rogrammable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMTcatalyzedcytosine methylation. Our study sets abasis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNAm ethylation.

Cyclic peptides are highly desired molecules not only for basic research but also for many biomedical and pharmacological applications. Due to their potentially superior physicochemical properties as compared to their linear counterparts, they are considered as ideal candidates for studying protein-protein interactions, among others. Most of the methods developed in recent years to prepare cyclic peptides focus either on a synthetic or a recombinant route. While the former provides access to diversified, noncanonical peptides, including unnatural and D-amino acid, for example, the latter can harness the power of genetic randomization to generate and select from large peptide libraries. Only few approaches have been reported to prepare semisynthetic macrocycles that would benefit from both the advantages associated with synthetic and genetically encoded parts. We describe in this chapter a chemo-enzymatic method to make semisynthetic cyclic peptides in vitro from two fragments using protein trans-splicing and bioorthogonal oxime ligation.

Methyl-CpG binding domain (MBD) proteins and ten-eleven-translocation (TET) dioxygenases are the readers and erasers of 5-methylcytosine (5mC), the central epigenetic mark of mammalian DNA. We employ light-activatable human TET1 controlled by a genetically encoded photocaged serine to enable in vivo kinetic studies of their interplay at the common substrate methylated cytosine–guanine (mCpG). We identify the multidomain reader MBD1 to negatively regulate TET1-catalyzed 5mC oxidation kinetics via its mCpG-binding MBD domain. However, we also identify the third Cys-x-x-Cys (CXXC3) domain of MBD1 to promote oxidation kinetics by TET1, dependent on its ability to bind nonmethylated CpG, the final product of TET-mediated mCpG oxidation and active demethylation. In contrast, we do not observe differences in TET1 regulation for MBD1 variants with or without the transcriptional repressor domain. Our approach reveals a complex, domain-dependent interplay of these readers and erasers of 5mC with different domain-specific contributions of MBD1 to the overall kinetics of TET1-catalyzed global 5mC oxidation kinetics that contribute to a better understanding of dynamic methylome shaping.

Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent...