Isolation of Human Genomic DNA Sequences with Expanded Nucleobase Selectivity

Abstract

We report the direct isolation of user-defined DNA sequences from the human genome with programmable selectivity for both canonical and epigenetic nucleobases. This is enabled by the use of engineered transcription-activator-like effectors (TALEs) as DNA major groove-binding probes in affinity enrichment. The approach provides the direct quantification of 5-methylcytosine (5mC) levels at single genomic nucleotide positions in a strand-specific manner. We demonstrate the simple, multiplexed typing of a variety of epigenetic cancer biomarker 5mC with custom TALE mixes. Compared to antibodies as the most widely used affinity probes for 5mC analysis, i.e., employed in the methylated DNA immunoprecipitation (MeDIP) protocol, TALEs provide superior sensitivity, resolution and technical ease. We engineer a range of size-reduced TALE repeats and establish full selectivity profiles for their binding to all five human cytosine nucleobases. These provide insights into their nucleobase recognition mechanisms and reveal the ability of TALEs to isolate genomic target sequences with selectivity for single 5-hydroxymethylcytosine and, in combination with sodium borohydride reduction, single 5-formylcytosine nucleobases.

Chemoselective labeling and site‐specific mapping of 5‐formylcytosine as a cellular nucleic acid modification

“…Although this traditional method provided early insights into the methylation status of CpG islands and was later coupled with deep sequencing, this approach is limited by the availability of selective restriction enzymes for diverse sequence contexts [42][43][44]. Immunoprecipitation with modification-specific antibodies allowed for enrichment prior to sequencing for analyzing the spatial distribution of epigenetic bases in the genome [36,37,[47][48][49], and transcription-activator-like effector (TALE)-based protein scaffolds were engineered to analyze 5mC and its oxidized derivatives with programmable sequence selectivity [50][51][52]. Immunoprecipitation with modification-specific antibodies allowed for enrichment prior to sequencing for analyzing the spatial distribution of epigenetic bases in the genome [36,37,[47][48][49], and transcription-activator-like effector (TALE)-based protein scaffolds were engineered to analyze 5mC and its oxidized derivatives with programmable sequence selectivity [50][51][52].…”

“…In addition, antibody-based detection methods, including dot blot analysis and immunofluorescence staining, were used to monitor 5mC and 5hmC in various tissues [45,46]. Immunoprecipitation with modification-specific antibodies allowed for enrichment prior to sequencing for analyzing the spatial distribution of epigenetic bases in the genome [36,37,[47][48][49], and transcription-activator-like effector (TALE)-based protein scaffolds were engineered to analyze 5mC and its oxidized derivatives with programmable sequence selectivity [50][51][52].…”

Recognition of Oxidized 5‐Methylcytosine Derivatives in DNA by Natural and Engineered Protein Scaffolds

“…The employment of TALEs with RVDs for sensing mC or hmC in affinity enrichment assays enabled the detection of the target nucleobases at single, user‐defined nucleotide positions in the zebrafish and human genomes. This occurred with strand‐selectivity and higher sensitivity than an antibody employed for the same targets ,. Natural and mutant TALE repeats were also analyzed for binding to the pro‐ and eukaryotic epigenetic nucleobase N6‐methyladenine (6 mA) and found to be highly promiscuous …”

“…This occurred with strand-selectivity and higher sensitivity than an antibody employed for the same targets. [130,131] Natural and mutant TALE repeats were also analyzed for binding to the pro-and eukaryotic epigenetic nucleobase N6-methyladenine (6 mA) [132][133][134] and found to be highly promiscuous. [135] Similar to antibodies, TALE proteins can be employed for staining DNA in live and fixed cells, but allow to do so in a sequence specific manner [113,[136][137][138][139] Combination of TALEs bearing RVDs with epigenetic nucleobase selectivity and fluorescent protein domains or effector domains thus has potential to develop applications not offered by approaches based on nucleobase conversions or proteins without programmable selectivity.…”

Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors

“…We have recently identified TALE repeats with selectivities in the context of cytosine 5-modifications [37][38][39]. These enabled the design of TALEs for the detection of epigenetic nucleobases at single, userdefined genomic positions by affinity enrichment [40]. By contrast, the interactions of natural and engineered TALE repeats with 4mC are unknown.…”