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22 results on '"Kubik, Grzegorz"'

3. Programmable Protein-DNA Crosslinking for the Direct Capture and Quantification of 5-Formylcytosine

Author

Gieß, Mario, Munoz-Lopez, Alvaro, Buchmuller, Benjamin, Kubik, Grzegorz, and Summerer, Daniel

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-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.

Published
2019
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21. Programmable and highly resolved in vitro detection of 5-methylcytosine by TALEs.

Author

Kubik G, Schmidt MJ, Penner JE, and Summerer D

Subjects
DNA metabolism, 5-Methylcytosine chemistry, DNA chemistry, In Vitro Techniques methods
Abstract

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. We report an mC-detection assay based on TALE control of DNA replication that displays unexpectedly strong mC-discrimination ability in vitro. The status and level of mC modification at single positions in oligonucleotides can be determined unambiguously by this assay, independently of the overall target sequence. Moreover, discrimination is reliably observed for positions bound by N-terminal and central regions of TALEs. This indicates the wide scope and robustness of the approach for highly resolved mC detection and enabled the detection of a single mC in a large, eukaryotic genome., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Published
2014
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22. Calcium-catalyzed cyclopropanation.

Author

Haven T, Kubik G, Haubenreisser S, and Niggemann M

Subjects
Catalysis, Cations chemistry, Nitrogen chemistry, Stereoisomerism, Thermodynamics, Calcium chemistry, Cyclopropanes chemistry
Published
2013
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