Cap-specific N6-adenosine methyltransferases and applications in m6A Cap-1 mRNA synthesis

In addition to the 5' Cap-1 structure, the initiating adenosine of mammalian mRNA can be further modified by PCIF1 (Phosphorylated C-terminal domain-interacting factor 1) to form 2'-O,N6-dimethyladenosine (m6A Cap-1). While the biological role is still under investigation, the implications of m6A Cap-1 in mRNA metabolism and viral-host interactions have spurred interest in PCIF1 and m6A Cap-1 as biomarkers, drug targets, and research tools. Here, we identify and characterize two PCIF1-like cap-specific N6-adenosine methyltransferases, PC04 and PC05 and evaluate their applicability in m6A Cap-1 mRNA synthesis in vitro. We found that both enzymes are highly specific to the m7G cap structure and highly active on Cap-1 RNA oligonucleotides and trinucleotide Cap-1 analogs at nanomolar concentrations. We demonstrate that PC04 or PC05 methylation reactions can be incorporated into established post-transcriptional capping and cap analog-based co-transcriptional in vitro transcription (IVT) workflows to produce m6A Cap-1 RNAs. Finally, we show that PC05 can be combined with a previously reported FCE::T7 RNA polymerase fusion to synthesize m6A Cap-1 mRNA from DNA templates in a single step, co-transcriptional manner. The robustness of the PCIF1-like cap N6-adenosine methyltransferases can accelerate research into the biological roles of cap m6A modification and development of highly effective mRNA-based therapeutics.