Orthologous mammalian A3A-mediated single-nucleotide resolution sequencing of DNA epigenetic modification 5-hydroxymethylcytosine.

Epigenetic modifications in genomes play a crucial role in regulating gene expression in mammals. Among these modifications, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are recognized as the fifth and sixth nucleobases in genomes, respectively, and are the two most significant epigenetic marks in mammals. 5hmC serves as both an intermediate in active DNA demethylation and a stable epigenetic modification involved in various biological processes. Analyzing the location of 5hmC is essential for understanding its functions. In this study, we introduce an orthologous mammalian A3A-mediated sequencing (OMA-seq) method for the quantitative detection of 5hmC in genomic DNA at single-nucleotide resolution. OMA-seq relies on the deamination properties of two naturally occurring mammalian A3A proteins: green monkey A3A (gmA3A) and dog A3A (dogA3A). The combined use of gmA3A and dogA3A effectively deaminates cytosine (C) and 5mC, but not 5hmC. As a result, the original C and 5mC in DNA are deaminated and read as thymine (T) during sequencing, while the original 5hmC remains unchanged and is read as C. Consequently, the remaining C in the sequence indicates the presence of original 5hmC. Using OMA-seq, we successfully quantified 5hmC in genomic DNA from lung cancer tissue and corresponding normal tissue. OMA-seq enables accurate and quantitative mapping of 5hmC at single-nucleotide resolution, utilizing a pioneering single-step deamination protocol that leverages the high specificity of natural deaminases. This approach eliminates the need for bisulfite conversion, DNA glycosylation, chemical oxidation, or screening of engineered protein variants, thereby streamlining the analysis of 5hmC. The utilization of orthologous enzymes for 5hmC detection expands the toolkit for epigenetic research, enabling the precise mapping of modified nucleosides and uncovering new insights into epigenetic regulation.