RNA Synthesis Kits

In vitro RNA synthesis requires a DNA template, RNA polymerase, NTPs and other factors. High-yield robust reactions require optimization of each reaction component. NEB offers five in vitro RNA synthesis kits, all of which have been optimized to generate reproducible yields of quality RNA.

For efficient translation to occur, most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5′ end and a poly(A) tail at the 3′ end. The HiScribe^®; T7 ARCA mRNA Synthesis Kit (NEB #E2060S) is designed to synthesize capped and tailed mRNAs for variety of applications. Capped mRNAs are synthesized by co-transcriptional incorporation of Anti-Reverse Cap Analog, ARCA, using T7 RNA Polymerase generating a Cap-0 structure. A poly(A) tail is then added by E. coli Poly(A) Polymerase. This kit is also available without E. coli Poly(A) Polymerase (NEB #E2065S) for use with DNA templates encoding a poly(A) stretch or not requiring a poly(A) tail. Both kits include DNase I and LiCl for DNA template removal and quick mRNA purification.

The Cap-1 structure has been reported to enhance mRNA translation efficiency (8) and hence may help improve expression in mRNA transfection and in microinjection experiments.  Cap-0 transcripts can be enzymatically converted to cap-1 in vitro. mRNA Cap 2 ́-O-Methyltransferase (NEB #M0366) adds a methyl group at the 2 ́-O position of the first nucleotide adjacent to the cap structure at the 5′ end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (Cap-0) resulting in a Cap-1 structure. Alternatively, Cap-1 mRNA can be synthesized co-transcriptionally with a trinucleotide cap analog such as CleanCap^â Reagent AG. The use of CleanCap reagent AG results in significant advantages over traditional dinucleotide co-transcriptional capping. CleanCap Reagent AG is a trinucleotide with a 5′-m7G joined by a 5′-5′ triphosphate linkage to an AG sequence. The adenine has a methyl group on the 2′-O position. The incorporation of this trinucleotide in the beginning of a transcript results in a Cap-1 structure.  The Hiscribe™ T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080S) is formatted with individual vials of NTPs and CleanCap Reagent AG to allow for partial or complete substitution of modified NTPs, with a total kit yield of 1.8 mg of mRNA.  Cap-1 mRNA synthesized from this kit is suitable for many applications, including transfections, microinjections, in vitro translation, preclinical mRNA therapeutic mRNA studies as well as RNA structure and function analysis.

Reference: Kuge, H., et al. (1998) Nucleic Acids Res, 26, 3208–3214.

CLEANCAP^® is a registered trademark of TriLink BioTechnologies, LLC.