Das, S. K.; Dutt, G.; Goswami, V.; Faiz, A.; joshi, A.

In vitro transcription (IVT) is a versatile procedure that facilitates template-directed synthesis of RNA molecules of any sequence, from short oligonucleotides to those of several kilobases, in quantities ranging from micrograms to milligrams. This technique, involving the engineering of a template with a bacteriophage promoter sequence, enables the synthesis of RNA for use in a variety of applications including structural studies, biochemical assays, and as functional molecules. Messenger RNA (mRNA) therapy has tremendous potential in regenerative medicine, disease treatment, and vaccination. Synthetic mRNA leverages the cell\'s natural translation machinery to produce proteins, with the ability to transfect cells and induce expression of target proteins under physiological conditions until it is eventually degraded. In this study, we explore the impact of pseudouridine ({Psi})-modified mRNA in enhancing RNA stability, translational efficiency, and immune compatibility for therapeutic use. By optimizing IVT conditions and employing cellulose-based purification techniques, we successfully synthesized high-quality, modified mRNA that demonstrated superior functional performance. Luciferase and GFP mRNA, synthesized with pseudouridine-modified rNTPs, exhibited improved stability, reduced immune activation, and enhanced translation efficiency in HEK293 cells. A sevenfold increase in luciferase activity and elevated GFP fluorescence confirmed the higher protein expression capabilities of the modified mRNA. Furthermore, cellulose bead purification effectively separated single-stranded RNA from double-stranded RNA contaminants, ensuring minimal immune response and maximizing transfection efficiency. These findings highlight the potential of pseudouridine-modified mRNA and refined purification methods for advancing mRNA-based therapies, from vaccines to protein-replacement treatments, setting the foundation for scalable, clinical-grade RNA production.