In vitro mRNA synthesis produces mRNA for vaccines and gene therapy. A linearized DNA template with a promoter (e.g., T7) is transcribed by T7 RNA polymerase using nucleotides, buffer, and magnesium ions. A 5' cap is added via a capping enzyme or cap analog, and a poly-A tail is included using the template or poly-A polymerase for stability. DNase I removes residual DNA, and the mRNA is purified. Key enzymes—BspQ1, T7 RNA polymerase, Pyrophosphatase, Inorganic, Murine RNase inhibitor, capping enzyme, poly-A polymerase, and DNase I—ensure functional mRNA.
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