Prepararea ARNm IVT

In vitro transcription (IVT) is the preferred method for preparing mRNA, capable of yielding micrograms to milligrams of mRNA on a laboratory scale. For research purposes, reagent suppliers have developed versatile IVT reaction systems suitable for mRNA of moderate length (1000-4000 nucleotides).

In this article, we will summarize in vitro transcription (IVT) of mRNA, including the conventional IVT reaction system used on a laboratory scale and the IVT reaction system suitable for ultra-long mRNA (>9000 nucleotides).

Kits enable co-transcriptional capping with CleanCap AG and reduce immunogenicity with m1ψ. DNase I removes residual DNA. Low yield can be due to poor mixing, reagent oxidation, or DNA template issues, which can be mitigated by centrifugation, fresh reagents, controls, and adjusting template amount/reaction time.

Yaohai Bio-Pharma provides various prefabricated IVT RNA products (liquid/freeze-dried powder) and LNP finished products, encoding proteins such as fluorescent proteins (eGFP, mCHERRY), luciferase, recombinase Cre, and antigen OVA, to meet different experimental or project needs.

IVT System for Ultra-long mRNA

Conventional IVT may not work for overly long mRNA (>9000 nt). A deep understanding and rational adjustment of IVT components are needed.

DNA Template: Linearized plasmids or PCR products with corresponding promoter sequences, dissolved in RNase-free H2O. A DNA template concentration higher than 40 nM is recommended for optimal IVT conditions.

DTT: Maintains enzyme activity during transcription. Fresh DTT should be added to the reaction buffer for optimal enzyme activity.

RNase Inhibitor: Inhibits RNase activity and maintains mRNA stability.

T7 RNA Polymerase: Catalyzes DNA transcription into RNA, corresponding to the promoter sequence. The activity of T7 RNA polymerase is affected by pH and magnesium ions.

Magnesium Ions (Mg2+): A cofactor for RNA polymerase. Too low concentrations reduce transcription efficiency, while too high concentrations lead to RNA degradation. Free [Mg2+] should be adjusted based on nucleotide concentration. Each nucleotide chelates one [Mg2+], so the [Mg2+] concentration should exceed the total nucleotide concentration.

Nucleoside Triphosphates (NTP): As substrates, NTPs are the basic units of RNA. NTP concentrations above 7 mM have a positive impact on mRNA production.

Spermidine: Stabilizes DNA-enzyme complexes and stimulates transcription reactions; however, excessive concentrations have inhibitory effects. The recommended spermidine concentration is between 1 and 3 mM.

Inorganic Pyrophosphatase (PPase): Added to IVT reactions to prevent the accumulation of inorganic pyrophosphate ions (PPi), avoiding their chelation with Mg2+ and ensuring sufficient free Mg2+.

Leveraging pioneering technology and IVT mRNA experience, Yaohai Bio-Pharma provides a one-stop IVT RNA service, encompassing sequence design, IVT RNA prep (with m1ψ mods), RNA cyclization, purification, freeze-drying, LNP encapsulation, and quality testing.

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