N 1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells

Abstract

Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stem from the high protein expression from m1Ψ containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1Ψ significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1Ψ modification have enormous potentials in the research and application of biofunctional RNA tools.

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

“…Modified nucleosides such as N1mΨ, Ψ, and 5mC have been widely used for mRNA transfection to prevent immune responses and increase translations [3][4][5]45 . Recently, we found that such nucleoside modifications also affect the affinities of RBPs including MS2CP 18 . Because the binding of RBPs near the 5′ terminus of mRNAs decreases translation 16 and because RBP affinities to the mRNAs correlate with translational repression efficiency 46 , we can estimate the relative affinity of MS2CP to each target mRNA with different nucleosides based on MS2CP-1xDmrA-mediated translational repression in the absence of A/C heterodimerizer (the condition in which DmrC-VPg(FCV) cannot interact with the target mRNAs, Fig.…”

“…In addition, we prepared these hmAG1 mRNA variants to have two different nucleoside compositions: N1-methyl-pseudouridine (N1mΨ) instead of uridine, and pseudouridine (Ψ) and 5-methylcytidine (5mC) instead of uridine and cytidine, respectively. Our recent study suggested that MS2CP could bind to N1mΨ-containing RNAs more strongly than to Ψ/5mC-containing RNAs 18 . These CaVT and hmAG1 mRNA variants were co-transfected into HeLa cells, and the hmAG1 fluorescence was measured by using a flow cytometer (Fig.…”

Synthetic modified messenger RNA for therapeutic applications

“…The replacement of natural U/C with Ψ/m 5 C ( Fig. 1 ) represented the best practice in the past, because they could reduce the immunogenicity of mRNA as well as increase its translation efficiency both in vitro and in vivo [ 61 , [63] , [64] , [65] , [66] ]. Ψ impedes stimulation of TLR7/8, PKR, OAS and RIG-I-mediated signaling pathways [ 12 , 62 , 67 ] ( Fig.…”

“…Other uridine analogs like 5-methyluridine (m 5 U), 5-methyoxy-uridine (mo 5 U), and 2-thiouridine (s 2 U) ( Fig. 1 ) could also reduce the mRNA immunogenicity, but find limited applications due to their lower translation performance than Ψ [ 36 , 48 , 65 , 72 , 81 ].…”

Allergic Reactions and Anaphylaxis to LNP-Based COVID-19 Vaccines

“…The synthetic mRNA is also least likely to instigate allergic responses per se, since the disclosed mRNA in the Pfizer-BioNTech COVID-19 vaccine contain N1-methylpseudouridine instead of uridine 9 to dampen immune responses and cytotoxicity induced by introducing mRNA into cells. 10 However, truncated and modified RNA trace impurities are present in the Pfizer-BioNTech COVID-19 vaccine; 9 thus, there is a possibility that aberrant proteins could be expressed and initiate delayed immunological reactions in some subjects.…”