Evolution of a reverse transcriptase to map N¹-methyladenosine in human messenger RNA

Chemical modifications to messenger RNA are increasingly recognized as a critical regulatory layer in the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile platform for directed evolution that rapidly selects for reverse transcriptases that install mutations at sites of a given type of RNA modification during reverse transcription, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N¹-methyladenosine (m¹A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m¹A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m¹A sites and revealed hundreds of m¹A sites in human mRNA. This work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods and datasets to study m¹A biology.