Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap® Analog by In Vitro Transcription.
Curr Protoc
; 1(2): e39, 2021 Feb.
Article
in En
| MEDLINE
| ID: mdl-33524237
ABSTRACT
Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap® AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap® AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1 IVT with CleanCap Basic Protocol 2 mRNA purification and analysis.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA Cap Analogs
/
RNA, Messenger
Type of study:
Guideline
Limits:
Humans
Language:
En
Journal:
Curr Protoc
Year:
2021
Document type:
Article