RNA: mRNA Capping by Vaccinia Capping enzyme

#mRNACapping is a necessary step both in vivo and in vitro, for efficient translation of #mRNA apart from stabilization and transport between cellular compartments in eukaryotic systems. The Vaccinia Capping Enzyme (#VCE) is a very efficient and robust capping system from the #Vaccinia virus and is scalable to #GMP level capping of mRNA.

Principle

The VCE has two subunits D1 and D12, D1 harboring enzymatic properties of #RNATriphopsphatase, #Guanylyltransferase and #GuanineMethyltransferase. The mRNA Cap 2’-O-Methyl transferase adds a methyl group at the 2'-O positions (Cap1) of the first nucleotide adjacent to the Cap structure. The #Cap1 structure reduces cellular innate response when the RNA is used in vivo.

Protocol

1. Denaturation of RNA: The in vitro transcribed #mRNA (of concentration ~2ug/uL) is denatured on a dry/ wet bath at 65C for 5 minutes followed by snap chill on ice for another 5 minutes.

2. The #RNA is then added to the following reaction mixture:

3. Incubate the reaction for 3 hours at 37C.

4. Purify the #cappedmRNA by #LithiumChloride precipitation method.

5. Check purity of capped mRNA by #SizeExclusionChromatography.

More to munch on:

# https://international.neb.com/products/m2080-vaccinia-capping-system#Product%20Information