Editing Cleancap mRNA Invitro Transcription (section)

== Enzymes for mRNA research ==

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986899/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986899/]

T7 RNA Polymerase 9135-130MCG 1

2598 pET29-Turbo-T7-RNAPo

E coli Inorganic Pyrophosphatase 9132-350MCG 1

2259 need to do cloning

DNase I 9136-70MCG 1&nbsp;

202 pET28-DNase1 need to subclone into pMBP vector

Ribonuclease Inhibitor 9134-180MCG 1

[http://18.206.161.119/eln/index.php/623_625_637_1494_1499_1734_1735_1740_1747_1750 pT7A30-RNaseInhibitor Sus scrofa (wild pig) RNase Inhibitor 1499]&nbsp;

Guanylyltransferase 9131-300MCG 2

2'-O-Methyltransferase 9130-1450MCG 2 An E. coli strain that carries the gene for the Vaccinia mRNA Cap 2´-O-Methyltransferase. Poly(A) Polymerase 9133-430MCG 2

Ribonuclease Inhibitor 9134-660MCG 2

Guanylyltransferase Catalyzes addition of 7-methylguanylate cap structure (Cap 0) to the 5’ end of RNA in three steps:


<div>T7 RNA Polymerase 2598 pET29-Turbo-T7-RNAPo</div><div>E coli Inorganic Pyrophosphatase&nbsp; 2259&nbsp;&nbsp;</div><div>Ribonuclease Inhibitor&nbsp; pT7A30-RNaseInhibitor Sus scrofa (wild pig)&nbsp; RNase Inhibitor 1499&nbsp;</div><div>Guanylyltransferase&nbsp; 2943</div><div>2'-O-Methyltransferase 2753</div><div>E coli&nbsp; Poly(A) Polymerase&nbsp; &nbsp; 2773</div><div>Yeast Poly(A) Polymerase&nbsp; 940 pET28b-yPolyA-Pol</div><div><br/></div><div><br/></div>
2’-O-Methyltransferase Reactions 1 & 2: Converts Cap 0 to Cap 1 by adding a methyl group to the ribose 2’-OH of the first nucleotide adjacent to the cap structure. Activity can be measured using the EPIgeneousTM Methyltransferase Assay (Cisbio).

Reaction 3: Conversion of an RNA substrate pppN(pN)n to GpppN(pN)n with increasing amounts of enzyme can be visualized by gel electrophoresis (uncapped, lower band; capped, upper band).

Methyl transfer from SAM to GpppN(pN)n producing SAH and Cap 0 mRNA can be measured using the EPIgeneousTM Methyltransferase Assay (Cisbio).

[https://www.aldevron.com/products/ivt-enzyme-kits https://www.aldevron.com/products/ivt-enzyme-kits]

IVT Buffer: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC306405/pdf/nar00265-0210.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC306405/pdf/nar00265-0210.pdf]

40 mM Tris-HCl (pH 8.1 at 37°C), 1 mM spermidine, 5 mM dithiothreitol, 50 ug/ml bovine serum albumin, 0.01% (vlv) Triton X-100 and 80 mg/ml polyethylene glycol (8000 MW)

[https://www.neb.com/gmp/gmp-grade-reagents-for-rna-synthesis https://www.neb.com/gmp/gmp-grade-reagents-for-rna-synthesis]

Inorganic pyrophosphatase [https://www.neb.com/products/m0361-pyrophosphatase-inorganic-e-coli https://www.neb.com/products/m0361-pyrophosphatase-inorganic-e-coli] PPase is prepared from an E. coli strain containing a clone of the E. coli inorganic pyrophosphatase gene.

DNaseI&nbsp;: [https://www.neb.com/products/m0303-dnase-i-rnase-free https://www.neb.com/products/m0303-dnase-i-rnase-free] An E. Coli strain that carries an MBP fusion clone of Bovine Pancreatic DNase I.