School of Engineering Department of Chemical and Biological Engineering 79 Engineering of Next Generation mRNA Vaccine Construct Supervisor: KUANG Becki Yi / CBE Student: CAO Jinhui / BIEN Course: UROP 1100, Spring The past several decades have witnessed the rapid progress of messenger RNA (mRNA) vaccines. During the COVID-19 pandemic, which facilitated the most expeditious vaccine development, mRNA vaccines have demonstrated great flexibility, promptness and safety. Despite the superiorities and advancements above, additional research that oriented on optimizing mRNA production via the widely-used in vitro transcription (IVT) method is required. This report will focus on the author’s UROP project with review of current mRNA production via IVT and introducing an innovative approach that employs site-specific terminal modification of linearized plasmids. It will also discuss the optimization process related to adjustments attempting to improve yield and purity of the IVT product. Engineering of Next Generation mRNA Vaccine Construct Supervisor: KUANG Becki Yi / CBE Student: CHANDRA Jones Edbert / BIOT Course: UROP 1100, Fall Increasing utilization of synthetic mRNA as a therapeutic tool highlighted its advantages over other nucleic acid-based therapeutics. Despite that, challenges such as mRNA instability, low expression level, and immune responses hinder broader applications. The study focuses on the synthesis of mRNA through in vitro transcription (IVT), and assessment of expression levels influenced by a synthetic microRNA switch. The successful synthesis of mRNA and expression analysis found that this synthetic mRNA design may potentially be broadly used to promote clinical applications. Recommendations on future directions are also emphasized to refine synthetic mRNA not only to improve stability but also to reduce immunogenicity. Engineering of Next Generation mRNA Vaccine Construct Supervisor: KUANG Becki Yi / CBE Student: LIYANARACHCHI Pasindu Nikil / BIEN Course: UROP 1100, Summer This project explores a synthetic mRNA construct designed to regulate protein translation in mammalian cells using RNA–protein interactions. The reporter mRNA, 3×MS2–eGFP–120A, contains MS2 stem-loop sequences in the 5’UTR that binds to the MS2 coat protein (MCP), which blocks translation. A literature review on RNA-based translational control and synthetic gene circuits informed the experimental design. The construct was assembled via fusion PCR by combining independently prepared 5’UTR, ORF, and 3’UTR fragments. After confirmation through agarose gel electrophoresis, the construct was transcribed into capped mRNA using in vitro transcription. The RNA was purified and introduced into HEK293 cells via reverse transfection. Confocal imaging showed that MCP co-transfection suppressed eGFP fluorescence, confirming protein-dependent control of translation in synthetic mRNA circuits.
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