Evolution of a Reverse Transcriptase to Map N(1)-Methyladenosine in Human mRNA

Chemical modifications on 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 directed evolution platform 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 N(1)-methyladenosine (m(1)A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m(1)A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m(1)A sites and revealed hundreds of m(1)A sites in human messenger RNA. Together, this work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods, and datasets to study m(1)A biology.