SFB 1309
Robust Bisulfite-Free Single-Molecule Real-Time Sequencing of Methyldeoxycytidine Based on a Novel hpTet3 Enzyme
2024-11-25
Hanife Sahin, Raheleh Salehi, Shariful Islam, Markus Müller, Pascal Giehr, and Thomas Carell
Angew. Chem. Int. Ed. 2024, 63, e202418500
In addition to the four canonical nucleosides dA, dG, dC and T, genomic DNA contains the additional base 5-methyldeoxycytidine (mdC). The presence of this methylated cytidine nucleoside in promoter
regions or gene bodies significantly affects the transcriptional activity of the corresponding gene. Consequently, the methylation patterns of genes are crucial for either silencing or activating genes. Sequencing the positions of mdC in the genome is therefore of paramount importance for early cancer diagnostics as it helps determine incorrect gene expression. Currently, the bisulfite method is the gold standard for mdC-sequencing. However, this method has the drawback that the majority of the input DNA is degraded during the bisulfite treatment. Additionally, bisulfite sequencing is prone to errors.
Here, we report a benign, bisulfite-free mdC sequencing method termed EMox-seq, which is based on third-generation single-molecule SMRT sequencing. The foundation of this technology is a new Tet3 enzyme that efficiently oxidizes mdCs to 5-carboxycytidine (cadC). In turn, cadC provides an excellent readout by SMRT sequencing using specially trained AI-based algorithms.
