|Authors:||Gieß, Mario David|
|Title:||Chemoselective conjugation strategies for the programmable detection of epigenetic cytosine 5-modifications with transcription activator-like effectors|
|Abstract:||Methylation at the cytosine (C) carbon-5 position in DNA is a reversible regulatory element of transcription in mammalian cells involved in development and disease. An active demethylation pathway through iterative oxidation of 5-methylcytosine (5mC) has been identified that leads to abasic sites at which unmodified C are restored via the base excision repair (BER) pathway. This process yields 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as partially stable intermediates. An increasing amount of data indicate that these 5-modified cytosines can also act as epigenetic regulatory elements and are involved in developmental and pathological processes. However, the function of epigenetic 5-modified cytosines are not fully understood and require sensitive typing and profiling approaches with high resolution. Engineered transcription activator-like effector (TALE) proteins have been established as programmable DNA-binders for the detection of epigenetic 5-modified cytosines. However, previous efforts to engineer new TALE selectivities did either not provide full selectivity for epigenetic 5-modified cytosines or had low affinity to target DNA. To overcome these hurdles, two novel TALE-based strategies based on chemical conjugation reactions were developed. In the first approach, a size-reduced TALE repeat with universal binding to the four canonical nucleobases and four epigenetic 5-modified cytosines was engineered. Chemoselective conjugation of 5hmC, 5fC or 5caC to dedicated blocking groups was found to abolish TALE binding. This enabled complete decoding of the three oxidized 5mC derivatives (ox5mCs) at single positions within target DNA. In the second approach, the non-canonical amino acid (ncAA) para-acetylphenylalanine (pAcF) was incorporated into dedicated positions of the universal TALE repeat. TALEs bearing pAcF maintained universal binding to cytosine 5-modified nucleobases. Furthermore, pAcF-bearing TALE repeats and 5fC-bearing target DNA could be crosslinked through oxime condensation via bifunctional hydroxylamine linkers. This enabled robust and selective enrichment of 5fC-bearing target DNA from genomic DNA (gDNA) backgrounds. Taken together, the addition of selective conjugation chemistries to TALE-based detection methods expand the toolbox for the selective, programmable analysis of epigenetic 5-modified cytosines.|
|Subject Headings:||Chemische Biologie|
|Subject Headings (RSWK):||Epigenetik|
|Appears in Collections:||Lehrstuhl für Chemische Biologie der Nukleinsäuren|
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