Harnessing pDNA Hydrogels for Cell-Free mRNA Synthesis

Author

Rachel A. Achong, University of New HampshireFollow

Date of Award

Spring 2024

Project Type

Senior Honors Thesis

College or School

COLSA

Department

Molecular, Cellular, and Biomedical Sciences

Program or Major

Biochemistry, Molecular, and Cellular Biology

Degree Name

Bachelor of Science

First Advisor

Nathan J. Oldenhuis

Abstract

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. The production of mRNA requires a plasmid DNA (pDNA) template. These two components are similar in chemical composition and properties, making their separation and the isolation of the mRNA difficult. 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 with the aim of generating gram-scale amounts of pDNA for cell-free mRNA synthesis. This protocol was utilized to generate pUCP20T-eYFP and pUCBB-pT7-eGFP to produce the fluorescent protein, eYFP and eGFP in initial solution cell-free synthesis trials in from existing, commercial cell-free synthesis kits with the aim of these preliminary trials to demonstrate the chosen plasmid’s level of compatibility of with the cell-free synthesis kit, providing a baseline proof of concept for its accessibility by cellular machinery. Though the initial trials have yet to exhibit cell-free synthesis activity, this work will lay the groundwork for future pDNA cell-free synthesis trials with the goal of scaling up to eventual cell-free mRNA synthesis with pDNA hydrogels.

Recommended Citation

Achong, Rachel A., "Harnessing pDNA Hydrogels for Cell-Free mRNA Synthesis" (2024). Honors Theses and Capstones. 818.
https://scholars.unh.edu/honors/818