5'-end capping is an essential modification of mRNA. mRNAs with cap structures, especially Cap1 structures, facilitate mRNAs evade innate immune responses in vivo, resulting in efficient protein translation.
Enzymatic capping (the two-step method) is the conventional method of mRNA capping, similar to the capping process in eukaryotic organisms. Under the action of a series of enzymes, 7-methylguanine (m7G) is linked to the 5'-end of mRNA through a 5'-5' triphosphate bond and undergoes methylation modification to form the cap structure Cap 1 (m7GpppN).
Diagram of natural cap structure formation
The enzymatic capping reaction flow is as follows:
Linearized plasmid DNA is used as a template for in vitro transcription (IVT) in the presence of T7 polymerase, and mRNA with a 5' end-cap structure is formed after a one-step purification using the vaccinia capping enzyme and 2'-O-methyltransferase.
Services Details
Optional Service | Service Details | Delivery Period (Day) |
mRNA enzymatic capping | Enzymatic capping reaction | 1 |
Capping reaction optimization - optional | Reaction component design and optimization | 3~7 |
Our Features
- Design and optimization of the capping reaction component
The capping reaction component is optimized, and the production of mRNA transcript is greatly increased.
- In vitro expression verification
The capped mRNA is transfected into 293T cells, and the expression of the target protein can be detected.
- Stringent control of RNase
Through stringent control of the RNaseson experimental environment and consumables, mRNA degradation is effectively prevented.
Case study
Yaohai Bio-Pharma’s mRNA platform has built a perfect capping reaction process.
For eGFP mRNA, an mRNA pre-product prepared by enzymatic capping, an eGFP fluorescence signal (green fluorescence) at a high level can be observed after transfecting 293T cells for 24 hours, which is detected by Western Blot (WB), demonstrating that the target protein enhanced green fluorescent protein (eGFP) can be efficiently expressed in vitro.
Expression of Enzymatic Capped eGFP mRNA in 293T Cell
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