Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine.
Chembiochem
; 22(4): 645-651, 2021 02 15.
Article
em En
| MEDLINE
| ID: mdl-32991020
ABSTRACT
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 (5â
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 5â
mC, and a control TALE targeting the position with a universal repeat that binds both C and 5â
mC. To enhance this approach, we report herein the development of novel 5â
mC-selective repeats and their integration into TALEs that can replace universal TALEs in imaging-based 5â
mC analysis, resulting in a methylation-dependent response of both TALEs. We screened a library of size-reduced repeats and identified several 5â
mC binders. Compared to the 5â
mC-binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5â
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 5â
mC functions in chromatin regulation by inâ
situ imaging with increased dynamic range.
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Base de dados:
MEDLINE
Assunto principal:
Processamento de Imagem Assistida por Computador
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Sequências Repetitivas de Ácido Nucleico
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Sondas Moleculares
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Metilação de DNA
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5-Metilcitosina
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Efetores Semelhantes a Ativadores de Transcrição
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article