Engineering of a DNA Polymerase for Direct m(6)A Sequencing

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N-6-methyladenosine (m(6)A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m(6)A-containing RNA prior to sequencing, since m(6)A modifications are generally erased during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m(6)A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N-6-methylation. We identified a mutant that exhibits increased misincorporation opposite m(6)A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m(6)A sites directly from the sequencing data of untreated RNA samples.