RNA Capping

RNA capping is an essential modification for biologically functional mRNA that can be generated using NEB’s RNA capping enzymes, synthetic cap analogs, modified nucleotides, methylase and RNA synthesis kits amenable to or designed for RNA Capping and Poly(A) Tailing.

In eukaryotic cells, most RNA capping enzymes add a 7-methylguanylate (7mG) structure to the 5´ end of nascent, pre-mRNA, a 5´ → 5´ triphosphate linkage. These modifications prevent RNA degradation, promote translation, and modulate its immunogenicity. Research and development objectives are best achieved with careful selection of the RNA capping approach considering comprehensive workflows and the desired properties for the capped RNA product.

RNA capping is relevant to many active areas of R&D including in vivo therapies, mRNA vaccines, CRISPR genome editing tools, and RNA inhibitor screening. RNA capping techniques integrate with cell-free protein synthesis, in situ experiments involving transfection or microinjection, generating pluripotent stem cells, or labeled RNA probes, and transcription RNA synthesis (IVT). Cap-1 RNA structure is desirable for increased biological activity and reduced immunogenicity in vivo, including mRNAs designed for protein synthesis

Guidance for generating Cap-1 mRNA

For IVT, ultimate mRNA yields, and workflow efficiencies are strongly influenced by choice of molecular strategies and the desired processing scale. Capping RNA in vitro can be performed in two ways; post-transcriptionally using capping enzymes, GTP and S-adenosyl methionine (SAM), or co-transcriptionally by including synthetic RNA cap analogs.

Cap-1 can be generated co-transcriptionally by including either dinucleotide RNA cap analogs, or the trinucleotide CleanCap^® reagent AG. for the highest yield and >95% capping efficiencies. The HiScribe^® T7 mRNA Kit with CleanCap^® Reagent AG (NEB #E2080) utilizes an optimized RNA synthesis formulation and trinucleotide cap analog technology for co-transcriptionally capping mRNAs that contain a natural Cap-1 structure in a single simplified reaction without compromising RNA yield.

With a post-translational approach all capped RNA structures are added in the proper orientation for translation, but the choice of RNA capping enzymes can be guided by the desired molecule and workflow conditions. NEB offers both conventional Vaccina Capping Enzyme (VCE) (NEB #M2080) and the Faustovirus Capping Enzyme (FCE) (NEB #M2081). FCE has the highest capping efficiency, even on difficult substrates, and a broader temperature activity range. The mRNA Cap 2´-O-Methyltransferase (NEB #M0366S) can be added to convert Cap-0 to Cap-1 in the same reaction with both enzymatic systems.

Overall, co-transcriptional capping of mRNA with template encoded poly(A) tails is recommended for most bench scale applications. Whereas enzymatic capping is the recommended approach for large scale or mRNA manufacturing. All RNA capping enzymes are available in bulk formats. Vaccinia Capping Enzyme and mRNA Cap 2´-O-Methyltransferase are also offered in GMP grade formats. GMP-grade Faustovirus capping enzyme will be available soon.

Guidance for generating Cap-1 mRNA for bench-scale and large-scale

RNA Synthesis Kits enable multiple RNA Capping approaches

The HiScribe^® line of RNA synthesis kits and reagents have robust, highly scalable and flexible protocol options for RNA capping and tailing workflows. RNA synthesis can be performed with biotin- or dye-modified nucleotides for internal labeling. Fully substituted synthesized RNA can be achieved for protein replacement of stem-cell differentiation objectives. Both T7 RNA Polymerase and the SP6 RNA Polymerase formats are available. Options for mRNA capping approaches with HiScribe kits include separate Faustovirus capping enzyme and Vaccinia capping enzyme, mRNA Cap analogs, and HiScribe kits including ARCA or CleanCap^® Reagent AG.