Overcoming conservation in TALE–DNA interactions: a minimal repeat scaffold enables selective recognition of an oxidized 5-methylcytosine

Maurer, Sara; Buchmuller, Benjamin; Ehrt, Christiane; Jasper, Julia; Koch, Oliver; Summerer, Daniel

doi:10.1039/c8sc01958d

Cited by 10 publications

(8 citation statements)

References 46 publications

(55 reference statements)

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“…By concatenating a series of repeats, one can build a programmable TALE for a desired sequence. Currently, an engineered TALE exists to differentiate 5mC, [32–34] 5hmC, [35,36] and 5caC [37,38] . While there is not yet a specific TALE available for 5fC, the Summerer group has developed an alternate strategy.…”

Section: Dna Modificationsmentioning

confidence: 99%

“…Currently, an engineered TALE exists to differentiate 5mC, [32][33][34] 5hmC, [35,36] and 5caC. [37,38] 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.…”

Section: Methyl Cytosine Sequencing and Detectionmentioning

confidence: 99%

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Advances and Opportunities in Epigenetic Chemical Biology

2020

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The study of epigenetics has greatly benefited from the development and application of various chemical biology approaches. In this review, we highlight the key targets for modulation and recent methods developed to enact such modulation. We discuss various chemical biology techniques to study DNA methylation and the post-translational modification of histones as well as their effect on gene expression. Additionally , we address the wealth of protein synthesis approaches to yield histones and nucleosomes bearing epigenetic modifications. Throughout, we highlight targets that present opportunities for the chemical biology community, as well as exciting new approaches that will provide additional insight into the roles of epigenetic marks.

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Section: Dna Modificationsmentioning

confidence: 99%

Section: Methyl Cytosine Sequencing and Detectionmentioning

confidence: 99%

Advances and Opportunities in Epigenetic Chemical Biology

2020

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“…We constructed a library of “size‐reduced” TALE repeats containing an RVD deletion, since this general approach has recently been particularly successful for the discovery of novel nucleobase selectivities [10,14d,e] . We deleted amino acid 13 and introduced a single random position at residue 12 that covered 11 amino acids with polar side chains.…”

Section: Figurementioning

confidence: 99%

“…TALE library generation : Library of NX* repeat modules for repeat position 5 (pNX*5) was generated as previously described [24] by restriction ligation using plasmid pHD5 as template. Briefly, pHD5 vector and respective annealed oligos (see the Supporting Information) were digested with NcoI and XhoI (New England Biolabs) and ligated with T4 DNA ligase.…”

Section: Figurementioning

confidence: 99%

Engineered TALE Repeats for Enhanced Imaging‐Based Analysis of Cellular 5‐Methylcytosine

et al. 2020

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Transcription‐activator‐like effectors (TALEs) are repeat‐based, programmable DNA‐binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging‐based analysis of cellular 5‐methylcytosine (5 mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co‐stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C‐selective repeat that is blocked by 5 mC, and a control TALE targeting the position with a universal repeat that binds both C and 5 mC. To enhance this approach, we report herein the development of novel 5 mC‐selective repeats and their integration into TALEs that can replace universal TALEs in imaging‐based 5 mC analysis, resulting in a methylation‐dependent response of both TALEs. We screened a library of size‐reduced repeats and identified several 5 mC binders. Compared to the 5 mC‐binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5 mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co‐stains of cellular SATIII DNA with a corresponding C‐selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5 mC functions in chromatin regulation by in situ imaging with increased dynamic range.

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“…23,25 The selectivity of TALE repeats is defined by the RVD, with the RVDs NI, NN, NG and HD binding A, G(A), T, and C, respectively. 24 TALE repeats with selectivity for 5mC and oxidized derivatives have been engineered, [27][28][29][30][31] and enabled detection at single, user-defined positions by affinity enrichment. 32 However, 5fC exhibits low genomic levels, 17 and no TALE repeats for its selective, sensitive detection have been reported.…”

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confidence: 99%

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

et al. 2019

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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.

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Overcoming conservation in TALE–DNA interactions: a minimal repeat scaffold enables selective recognition of an oxidized 5-methylcytosine

Abstract: Transcription-activator-like effectors (TALEs) have been engineered to selectively recognize the epigenetic nucleobase 5-carboxylcytosine.

Cited by 10 publications

References 46 publications

Advances and Opportunities in Epigenetic Chemical Biology

Advances and Opportunities in Epigenetic Chemical Biology

Engineered TALE Repeats for Enhanced Imaging‐Based Analysis of Cellular 5‐Methylcytosine

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

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