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Enzymes for mRNA Production

Enzymes for mRNA Production

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Modality

Enzymes for RNA Production

Key Enzymes for Long-RNA Production

mRNA, as a type of long coding RNA, performed safely and efficiently in a broader range of people during COVID-19. Other long-coding RNAs, like self-amplifying RNA (saRNA) and circular mRNA (circRNA) also showed efficiency in pre-clinical and clinical studies.

Large scale production of long-coding RNA relies on in vitro transcription (IVT) by several enzymatic reactions. The steps of RNA production include template DNA preparation, IVT RNA synthesis, circulation, DNA removal, RNA modification (capping and tailing), and RNA purification.

There are several key enzymes used in long coding RNA production (e.g., mRNA, saRNA, and circRNA), including restriction endonucleases, T7 RNA polymerase (T7 RNAP), RNase inhibitor, inorganic pyrophosphatase, DNase I, vaccinia capping enzyme and RNase R.

Process

Enzyme

Function

Template DNA preparation

Restriction endonucleases

Linearization of plasmid DNA (pDNA) to avoid generating longer transcript.

IVT RNA synthesis

T7 RNA polymerase (T7 RNAP)

Bind to the T7 promoter and generate specific RNA transcript; Play a key role during IVT reaction.

RNase Inhibitor, recombinant

Inhibit RNase activity during IVT reaction.

Pyrophosphatase, Inorganic (iPPase)

Prevent pyrophosphate during IVT reaction.

DNA removal

DNase I

Remove the DNA template.

RNA modification

Vaccinia capping enzyme

Add Cap1 structures to the 5′ ends of IVT mRNA.

2’-O-Methyltransferas

circRNA enrichment

RNase R

Digest linear RNA and enrich circular RNA.
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