Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.
5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein-DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single-base resolution. We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to induce T-to-C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T-to-C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU-rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.
['Kawasaki|Fumiko|F|0000-0002-6641-2599', 'Martínez Cuesta|Sergio|S|0000-0001-9806-2805', 'Beraldi|Dario|D|0000-0003-1504-5212', 'Mahtey|Areeb|A|0000-0002-4509-5272', 'Hardisty|Robyn E|RE|0000-0002-9900-1155', 'Carrington|Mark|M|0000-0002-6435-7266', 'Balasubramanian|Shankar|S|0000-0002-0281-5815']
5-hydroxymethyluracil 5-formyluracil DNA epigenetics thymine modifications