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

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 (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.

SUBMITTER: Munoz-Lopez A

PROVIDER: S-EPMC7894354 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Muñoz-López Álvaro Á Jung Anne A Buchmuller Benjamin B Wolffgramm Jan J Maurer Sara S Witte Anna A Summerer Daniel D

Chembiochem : a European journal of chemical biology 20201106 4

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 rep ...[more]

PMID: 32991020

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Project description:The use of contrast agents in breast imaging has the capability of enhancing nodule detectability and providing physiological information. Accordingly, there has been a growing trend toward using iodine as a contrast medium in digital mammography (DM) and digital breast tomosynthesis (DBT). Widespread use raises concerns about the best way to use iodine in DM and DBT, and thus a comparison is necessary to evaluate typical iodine-enhanced imaging methods. This study used a task-based observer model to determine the optimal imaging approach by analyzing six imaging paradigms in terms of their ability to resolve iodine at a given dose: unsubtracted mammography and tomosynthesis, temporal subtraction mammography and tomosynthesis, and dual energy subtraction mammography and tomosynthesis.Imaging performance was characterized using a detectability index d', derived from the system task transfer function (TTF), an imaging task, iodine signal difference, and the noise power spectrum (NPS). The task modeled a 10 mm diameter lesion containing iodine concentrations between 2.1 mg/cc and 8.6 mg/cc. TTF was obtained using an edge phantom, and the NPS was measured over several exposure levels, energies, and target-ﬁlter combinations. Using a structured CIRS phantom, d' was generated as a function of dose and iodine concentration.For all iodine concentrations and dose, temporal subtraction techniques for mammography and tomosynthesis yielded the highest d', while dual energy techniques for both modalities demonstrated the next best performance. Unsubtracted imaging resulted in the lowest d' values for both modalities, with unsubtracted mammography performing the worst out of all six paradigms.At any dose, temporal subtraction imaging provides the greatest detectability, with temporally subtracted DBT performing the highest. The authors attribute the successful performance to excellent cancellation of inplane structures and improved signal difference in the lesion.

Dataset Information

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

Publications

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

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