IVT Enzymes for mRNA

Guanylyltransferase 

Guanyltransferase (GT), also known as Vaccinia Capping Enzyme (VCE), catalyzes addition of 7-methylguanylate cap structures (Cap 0) to the 5’ end of RNA in three steps:

Reactions 1 & 2: Conversion of an RNA substrate pppN(pN)n to GpppN(pN)n with increasing amounts of enzyme was visualized by gel electrophoresis (uncapped, lower band; capped, upper band). One unit (U) is the amount of enzyme activity that caps 50% of a 5’-ppp oligo in 30 minutes at 37°C.

Reaction 3: Methyl transfer from SAM to GpppN(pN)n producing SAH and cap-0 mRNA was measured using the EPIgeneousTM Methyltransferase Assay (Cisbio). One unit (U) is the amount of enzyme activity that produces 1 pmol of SAH from SAM in 30 minutes at 37°C using cap-0 substrate.

2’-O-Methyltransferase

2'-O-Methyltransferase adds a methyl group to the 2'-OH of the first nucleotide adjacent to the m7Gppp5'N cap. 2'-O-Methyltransferase utilizes
S-adenosylmethionine as the methyl group donor.

T7 RNA Polymerase

T7 RNA Polymerase catalyzes synthesis of RNA in the presence of a DNA template containing the T7 phage promoter.

Inorganic Pyrophosphatase

Inorganic Pyrophosphatase catalyzes hydrolysis of inorganic pyrophosphate, producing two molecules of phosphate.

Ribonuclease Inhibitor

Ribonuclease Inhibitor is a potent, noncompetitive inhibitor of pancreatic-type ribonucleases including ribonucleases A, B and C.

DNase I

DNase I nonspecifically cleaves single- or double-stranded DNA, producing di-, tri- and oligonucleotide products. DNase I is free of detectable ribonuclease activity.

Poly(A) Polymerase

Poly(A) Polymerase catalyzes addition of AMP from ATP to the 3' end of RNA in a template-independent manner.