Double-stranded RNA (dsRNA) is a critical by-product of wildtype T7 RNA Polymerase-based in vitro transcription. It needs to be minimized in final RNA preparations to reduce the risk of undesired immune responses[1-3]. Removal of such dsRNA impurities is typically performed by time-consuming & multi-step chromatographic processes[3-4].
HighYield T7 PURE RNA Synthesis Kit - based on a modified T7 RNA Polymerase (T7 PURE) – facilitates efficient RNA synthesis with reduced dsRNA formation (Fig. 1) thereby greatly streamlining down-stream purification protocols. Yields are comparable to wildtype T7 RNA Polymerase Mix after 2 h incubation at 37 °C (Tab. 1).
The kit is therefore ideally suited for RNA preparations where the dsRNA impurity level is critical for down-stream applications e.g. mRNA preparation for cell culture experiments.
Figure 1: HighYield T7 PURE RNA Polymerase Mix reduces dsRNA formation during in vitro transcription (IVT) compared to wildtype HighYield T7 RNA Polymerase Mix.
IVT conditions: 1 µg DNA template (1400 nt RNA transcript), 7.5 mM each unmodified NTPs (100 % UTP) or UTP fully replaced by N1-Methylpseudo-UTP (100 % me1Ψ-UTP), 1x HighYield T7 Reaction Buffer, 2 µl each wildtype HighYield T7 (T7 WT Mix), HighYield T7 PURE (T7 PURE Mix) or HighYield T7 P&L RNA Polymerase Mix (T7 P&L Mix). Incubation for 2 h at 37 °C.
Dot Blot Assay: Various amounts of in vitro transcribed, silica-membrane purified RNA (1400 nt) spotted on a nylon membrane. dsRNA detection with mouse J2 antibody & AP-conjugated secondary antibody.
Positive control: 142 nt dsRNA / Negative control: Poly(A) ssRNA
| 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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[1] Nelson et al. (2020) Impact of mRNA chemistry and manufacturing process on innate immune activation. Sci. Adv. 6:eaaz6893.
[2] Mu et al. (2018) An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res. 46 (10):5239.
[3] Karikó et al. (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 39 (21):e142.
[4] Baiersdörfer et al. (2019) A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA. Ther. Nucleic Acids 15:26.
[5] Guillerez et al. (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance. Natl. Acad. Sci. U.S.A 102:5958.
[6] Salvail-Lacoste et al. (2018) Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags. RNA 19:1003.
[7] Lyon et al. (2018) A T7 RNA Polymerase Mutant Enhances the Yield of 5'-Thienoguanosine-Initiated RNAs. ChemBioChem 19:142.
