The cap structure is added to the mRNA by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA) (NEB#S1411) using T7 RNA Polymerase. Poly(A) tail is incorporated during the transcription reaction. The kit also includes DNase I and LiCl for DNA template removal and quick mRNA purification.
Additionally, the kit is capable of partial incorporation of modified UTP and CTP (up to 50% each) without affecting the mRNA yield significantly. By using a DNA template encoding a poly(A) tail, capped and tailed modified mRNA can be synthesized in a single reaction in 30 minutes. mRNAs synthesized with the kit can be used for cell transfection, microinjection, in vitro translation and RNA vaccines.
ARCA is incorporated into mRNA exclusively in the correct orientation, generating capped mRNA that is more efficiently translated.
Standard cap analogs can be incorporated in either direction resulting in only 50% of capped mRNA that is functional in protein translation.