Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.
Angewandte Chemie (International ed. in English)
John Wiley & Sons Ltd.
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Kawasaki, F., Martínez Cuesta, S., Beraldi, D., Mahtey, A., Hardisty, R. E., Carrington, M., & Balasubramanian, S. (2018). Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.. Angewandte Chemie (International ed. in English), 57 (31), 9694-9696. https://doi.org/10.1002/anie.201804046
5-hydroxymethyluracil (5hmU) is formed via oxidation of thymine both enzymatically and non-enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as proteinDNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Here, 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, thus will contribute to promote the understanding of this modified base.
Wellcome Trust Herchel Smith Funds University of Cambridge
Wellcome Trust (099232/Z/12/Z)
Cancer Research UK (CB4330)
External DOI: https://doi.org/10.1002/anie.201804046
This record's URL: https://www.repository.cam.ac.uk/handle/1810/283401
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/