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

• Published in: Chembiochem : a European journal of chemical biology 2021-02, Vol.22 (4), p.645-651
• Main Authors: Muñoz‐López, Álvaro, Jung, Anne, Buchmuller, Benjamin, Wolffgramm, Jan, Maurer, Sara, Witte, Anna, Summerer, Daniel
• Format: Article
• Language: English
• Subjects: Bases (nucleic acids); Binders; Binding; Chromatin; Communication; Communications; Cytosine; Deoxyribonucleic acid; DNA; DNA methylation; epigenetics; Fluorescence; Gene sequencing; Imaging; imaging probes; membrane-less organelles; Nucleotide sequence; Selectivity; Transcription activation
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202000563

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. Two‐TALE test: Engineered transcription‐activator‐like effector (TALE) repeats can be used to design programmable receptors for improved in situ imaging‐based analysis of 5 mC in user‐defined DNA sequences. Pairs of natural 5 mC‐discriminating and engineered 5 mC‐binding TALEs allow nucleotide resolution analysis of 5 mC with response of both TALEs. This provides an impulse for the design of programmable imaging probes for studying 5 mC functions in chromatin regulation with increased dynamic range.