Title:

Harnessing pDNA Hydrogels for Cell-Free mRNA Synthesis

Vaccines are instrumental in protecting against human disease and improving public health. The demand for better techniques to mass-produce mRNA has been amplified through the publicized development of the SARS-CoV-2 (COVID-19) vaccine. A major bottleneck in the production of mRNA vaccines is the purification from its template plasmid DNA (pDNA) sequence. Incorporating the pDNA template into a chemically cross-linked hydrogel for use in cell-free synthesis would keep it out of solution, simplifying the subsequent purification. Unfortunately, current production and purification processes do not meet the supply demand needed to make pDNA hydrogels. Through a combination of academic and industry procedures, we optimized a scalable pDNA production and purification process through fed-batch fermentation and anion-exchange chromatography to generate gram-scale amounts of pDNA. This protocol was utilized to generate pDNA containing the template for our mRNA in the cell-free synthesis of eYFP from pUCP20T-eYFP. Initial cell-free synthesis trials using pUCP20T-eYFP and myTXTL Sigma70 master mix have not been successful but is likely due to incompatibility issues with our plasmid and cell-free synthesis kit. Future work will focus on optimization of solution cell-free synthesis before moving on to performing cell-free synthesis with chemically cross-linked pDNA hydrogels.

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