Isolation of genomic DNA sequences with expanded nucleobase selectivity using Transcription Activator-Like Effector Proteins

In mammals, DNA methylation is an epigenetic modification and the most widely characterized epigenetic nucleobase is 5-methylcytosine (5mC), that causes downregulation of gene transcription. Several studies have shown a direct link between the presence of 5mC in promoters of tumour suppressor genes and various cancers, autoimmune diseases, metabolic disorders and neurodegenerative ailments such as Schizophrenia, Fragile X and Prader-Willi syndrome. This thesis was focussed on the development of a novel approach for sequence specific detection of epigenetic C modifications in genomic DNA (gDNA) using Transcription Activator-Like Effector (TALE) proteins which are DNA binding proteins composed of repeats containing conserved amino acid sequences. The 12th and the 13th amino acid of Abstract 16 each repeat denoted as Repeat Variable Di-residue (RVD) however differ depending on the nucleobase targeted. TALE RVD HD (histidine aspartate) can discriminate between DNA sequences containing a C or 5mC at a single position. Identification of engineered RVDs capable of selectively recognizing other C modifications like hydroxymethyl-, formyl- and carboxylcytosine (5hmC, 5fC and 5caC) have also been reported. TALEs were employed in an affinity enrichment assay for modification sensitive binding and isolation of user-defined DNA sequence directly from gDNA samples. TALEs were employed in an affinity enrichment assay for modification sensitive binding and isolation of user-defined DNA sequence directly from gDNA samples. In combination with chemical reduction of 5fC to 5hmC, TALEs with a size-reduced RVDs were shown to successfully detect and discriminate between genomic sequence containing a 5hmC or 5fC. Successful discrimination of C, 5mC and N4-methylcytosine (4mC) using the established affinity enrichment assay further highlighted the use of TALEs as tool for studying the individual roles of various epigenetically modified nucleobases found in a genome. To enhance TALE binding sensitivity, positively charged amino acids were exchanged with alanine. Compared to non-modified TALEs, those with alanine mutations exhibited significantly increased 5mC sensitivity in affinity enrichment assay and in C selective transcription activation in HEK293T cells as demonstrated by the luciferase assay