In vitro synthesis of RNA (> 100 nt up to several thousand nt) is catalyzed by bacteriophage RNA polymerases using linear double-stranded DNA (dsDNA) as template (in vitro transcription).
Wildtype T7 RNA polymerase is the most efficient and widely used RNA polymerase however, challenges persist e.g. heterogeneity at the 3'-and 5'-end of RNA products as well as and high level of double-stranded RNA (dsRNA) by-products [1-8]. These challenges can be addressed by modified T7 RNA Polymerases that are available in a convenient ready-to-use kit configuration (Tab. 1).
The biological functionality of in vitro transcribed mRNA can be further increased by 5'-capping and nucleoside base modifications which significantly increase translation efficiency while reducing immunogenicity [9-24]. Such modifications are conveniently introduced by correspondingly modified nucleotides via T7 RNA polymerase-mediated in vitro transcription (Tab. 2). The optimal combination of 5’ capping and nucleoside base modification(s) however, needs to be individually determined for each mRNA target. Initial comparative studies can conveniently be performed with our HighYield T7 mRNA Testkits: HighYield T7 mRNA Modification Testkit.
| Product | Features | Yield after 30 min at 37 °C* | Yield after 2 h at 37 °C* |
|---|---|---|---|
| HighYield T7 RNA Synthesis Kit |
|
140 -160 µg | 140 -160 µg |
| HighYield T7 P&L RNA Synthesis Kit |
|
140 -160 µg | 140 -160 µg |
| HighYield T7 PURE RNA Synthesis Kit |
|
100 -120 µg | 140 -160 µg |
| HighYield T7 Enzyme Testkit |
|
||
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[15] Li et al.(2016) Effects of Chemically Modified Messenger RNA on Protein Expression. Bioconjugate Chem.27:849.
[16] Arango et al.(2018) Acetylation of Cytidine in mRNA Promotes Translation Efficiency. Cell175(7):1872.
[17] Sinclair et al.(2017) Profiling Cytidine Acetylation with Specific Affinity and Reactivity. ACS Chem. Neurosci.12(12):2922.
[18] Dominissini et al.(2016) The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA. Nature530:441.
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