Takara PrimeCap™ T7 RNA Polymerase (low dsRNA), 20,000 U

PrimeCap T7 RNA Polymerase (low dsRNA) is a DNA-dependent RNA polymerase that produces high yields of capped, in vitro-transcribed RNA with extremely low dsRNA byproduct. PrimeCap T7 RNA Polymerase (200 U/μl) and 10X T7 RNA Polymerase Buffer are included.

PrimeCap T7 RNA Polymerase (low dsRNA) produces single-stranded mRNA with very low dsRNA byproduct without sacrificing mRNA yield.T7 RNA Polymerase ver.2.0 (Cat. #2541A) and PrimeCap T7 RNA Polymerase were used to synthesize mRNA from the Positive Control Template (FLuc) plasmid (sold as part ofCat. #6144). CleanCap Reagent AG (4 mm; TriLink BioTechnologies) and Pyrophosphatase (Cat. #2450A/B) were added to the in vitro transcription reaction.Panel A. mRNA yields of using T7 RNA Polymerase ver.2.0 or PrimeCap T7 RNA Polymerase in a 20 µl reaction volume.Panel B. Percentage of dsRNA per reaction as compared to T7 RNA Polymerase ver.2.0 (WT on y-axis), as determined by a dsRNA ELISA kit.

Quality of in vitro-transcribed mRNAs produced with PrimeCap T7 RNA Polymerase (low dsRNA). FLuc mRNA was synthesized using Primecap T7 RNA (low dsRNA) or T7 RNA Polymerase ver.2.0 (Cat. #2541A) with CleanCap Reagent AG (3′OMe) in the absence (-) or presence (+) of N1-Methylpseudouridine-5′-Triphosphate (m1ΨTP). 1 ng of the resulting in vitro-transcribed mRNA was analyzed using an Agilent Bioanalyzer.Panel A. The bioanalyzer gel image shows a single band for mRNA produced using PrimeCap T7 RNA Polymerase (low dsRNA) and T7 RNA Polymerase ver.2.0.Panel B. Bioanalyzer electropherograms of mRNA produced using PrimeCap T7 RNA Polymerase (low dsRNA) or T7 RNA Polymerase ver.2.0 both show a single peak at the expected size without traces of major byproducts.

Effect of CleanCap Reagent AG concentration on mRNA yield, capping efficiency, and FLuc protein expression.FLuc mRNA was synthesized using PrimeCap T7 RNA Polymerase (low dsRNA) or T7 RNA Polymerase ver.2.0 (Cat. #2541A) with 4 mm or 8 mm CleanCap Reagent AG (3′OMe). The resulting mRNA was transfected into HEK 293T cells and FLuc protein expression was determined by a luciferase assay.Panel A. High yields (≥198 μg in a 20 μl reaction) were observed at both concentrations of CleanCap Reagent AG for both Prime T7 RNA Polymerase (low dsRNA) and T7 RNA Polymerase ver.2.0 and PrimeCap T7 RNA Polymerase. PrimeCap T7 RNA Polymerase exhibited higher capping efficiency (Panel B) and FLuc protein expression (Panel C) than T7 RNA Polymerase ver.2.0, even with only 4 mm of CleanCap Reagent AG.

Evaluation of in vitro-transcribed mRNA produced with PrimeCap T7 RNA Polymerase (low dsRNA) using different template lengths. mRNAs were synthesized using DNA templates ranging from 1–12.3 kb using PrimeCap T7 RNA Polymerase (low dsRNA) or T7 RNA Polymerase ver.2. (Cat. #2541A) with 4 mm of CleanCap Reagent AG (3′OMe). 200 ng of the resulting in vitro-transcribed mRNA from each reaction was analyzed by formaldehyde gel electrophoresis. Both PrimeCap T7 RNA Polymerase (low dsRNA) and T7 RNA polymerase ver.2.0 were capable of synthesizing the mRNAs of different lengths. The efficiency of the in vitro transcription reactions decreased with transcripts longer than 4.5 kb.