The vast majority of eukaryotic mRNA 5' end m7G cap structures improve RNA stability and significantly enhance translation. Anti-Reverse Cap Analog (ARCA) is used for co-transcription capping of mRNA and producing cap transcripts that are modified to ensure only positive incorporation since ARCA. Conventional cap analogues have two 3 '-OH groups, from which RNA polymerases can initiate transcription, while the 3 '-OH group close to m7G in ARCA is replaced by -OCH3 (as shown in the figure above), RNA polymerases can only use the remaining 3'-OH groups, forcing ARCA to be positively incorporated to produce 100% translatable cap RNA. ARCA can be used in combination with our modified nucleoside triphosphate (Nl-Me-pUTP, pUTP). By following the conditions in this note, the capping rate can reach approximately 80% and the crude transcript can reach 2-2.5 mg/ml.

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