PCR-generated DNA templates enable efficient, rapid, and cost-effective mRNA synthesis.

In vitro transcription (IVT) is a widely used technique for mRNA synthesis in both basic research and the development mRNA-based vaccines and therapies. The efficiency of IVT critically depends on the quality and integrity of the linear DNA templates. The conventional method for template DNA preparation involves plasmid propagation in bacteria followed by enzymatic linearization, which is labor-intensive and costly. Here, we describe a cell-free, PCR-based approach for generating high-quality, high-yield linear DNA templates. We extensively compared the PCR-based method with the conventional plasmid-based approach in terms of IVT efficiency, mRNA production, and the immunogenicity of the resulting mRNA-LNP (lipid nanoparticle) vaccines. Compared to the plasmid-derived DNA, the PCR-based method yielded higher amounts of both DNA templates and transcribed mRNA, while maintaining mRNA quality and integrity. Importantly, mRNA-LNP vaccines encoding the SARS-CoV-2 spike protein, generated from both methods, elicited robust and comparable immune responses in mice, with no significant differences observed between the two template methods. Our findings highlight the advantages of PCR-generated DNA templates as a rapid, efficient, and cost-effective alternative for mRNA synthesis, with broad applications in vaccine and therapeutic development.