Novel trinucleotide mRNA capping reagents: improved synthetic route and efficient cotranscriptional incorporation in...

The 5'-N ⁷-methylated guanosine triphosphate cap structure plays a critical role in mRNA translation and mRNA stability. The recent invention of cotranscriptional capping of mRNAs using trinucleotide capped primers (TCPs) allows for development of large-scale in vitro transcription (IVT) synthesis of mRNA carrying a eukaryotic Cap 1 structure (TCP-mRNA). Here we present a novel "one-pot-two-step" methodology for the synthesis of TCPs that improves the yield and simplifies the isolation and purification of the TCPs. Over 70 different modified TCPs, the analogs of a ^7mGpppA_mpG trimer, were synthesized, characterized, and tested for their ability to initiate IVT reaction. The results demonstrate that full complementarity of TCP to a template strand of dsDNA template at transcription initiation (start) site, at positions +1 and +2, is required and sufficient to obtain capped TCP-mRNA with high capping efficiency (>98%) and high yield (>5 mg/mL). This approach can be applied from small- to large-scale mRNA synthesis carrying various 5'-cap structures.