Multicistronic IVT mRNA for simultaneous expression of multiple fluorescent proteins

Synthetic mRNA has emerged as a promising gene expression system for more rapid and controllable induction of target proteins, due to no need of transnuclear localization. It can efficiently generate large amount of encoded proteins in cells as compared to that of conventional pDNA. In addition, the development of in vitro transcription (IVT) technique has enabled the facile modulation of synthetic mRNA's biochemical properties, further strengthening its applications in pharmaceutical sciences and engineering. In this study, we have investigated the IVT mRNA for simultaneous expression of three distinctive fluorescent (GFP, RFP, and BFP) proteins. To achieve simultaneous expression of the multiple proteins by a single platform, 2A peptides encoding sequences were used in the design of GRB mRNA. Self-cleavages of 2A peptides in the translated fusion proteins were confirmed by western blot analysis. The expression level of the IVT mRNA was optimized by evaluating the use of chemical modification, the method of poly A tailing, and the capping conditions of IVT process.