EGFP mRNA (N1-Methylpseudo-UTP modified)

Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methylcytidine to replace uridine and cytidine to avoid immune responses and cytotoxicity caused by the introduction of mRNA into cells. However, the effect of base modifications on the functionality of RNA tools is still unclear. This study found that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) instead of uridine performed significantly better than all other modified bases studied, showing higher microRNA and protein sensitivity, better cell type separation ability, and relatively low immunostimulation. The researchers found that the observed phenomenon originated from the high protein expression of mRNA containing m1Ψ and the effective translation inhibition in the presence of target microRNA or protein. In addition, synthetic gene circuits containing m1Ψ significantly improved the performance in cells. These results show that synthetic mRNA containing m1Ψ modification has great potential in the research and application of biological functional RNA tools.

In both 293FT and HeLa cells, EGFP expression was higher in miR-21 or MS2CP-sensing switches containing m1Ψ than in switches containing other modified bases. Similarly, the researchers found that EGFP mRNA containing m1Ψ had the highest expression level in HeLa cells, confirming that m1Ψ can enhance mRNA translation (Figure 1A). The researchers then examined the cellular degradation rate and transfection efficiency of the differently modified EGFP mRNAs and found no significant differences between the mRNAs, suggesting that the enhancement was not due to these parameters. Previous studies have shown that immune response is a key factor affecting the translation level of mRNA containing Ψ and m1Ψ. Here, the researchers co-transfected EGFP mRNA with EKB mRNA. This has been reported to minimize immune responses caused by nucleic acid transfection. An equal amount of control mRNA (encoding an inert bacterial protein (puromycin resistance protein; puroR)) was also co-transfected with EGFP mRNA as a control. Figure 1B shows that m1Ψ mRNA outperformed native mRNA and Ψ-containing mRNA in terms of EGFP expression even in the presence of EKB co-transfection. Although the increase in basal expression of m1Ψ-mRNA is not solely due to the attenuation of the innate immune response, these results clearly indicate that m1Ψ-mRNA can increase basal expression and enhance on-expression of the mRNA switch.

Figure 1. (A) Relative EGFP expression in HeLa cells transfected with EGFP mRNAs that carry different base modifications. (B) Relative EGFP expression in HeLa cells transfected with EGFP mRNAs with co-transfection of immune-evasive EKB mRNA or control mRNA. (Parr C J C, et al., 2020)