Engineering of a DNA Polymerase for Direct m⁶A Sequencing

Abstract

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N⁶-methyladenosine (m⁶A) 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 m⁶A-containing RNA prior to sequencing, since m⁶A 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 m⁶A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N⁶-methylation. We identified a mutant that exhibits increased misincorporation opposite m⁶A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m⁶A sites directly from the sequencing data of untreated RNA samples.