N1 methylpseudouridine 5 triphosphate m1ψtp

The DNA template for the mRNA vaccine was a DNA fragment encoding the HA protein of the influenza A virus (A/Puerto Rico/8/1934). DNA templates of the mRNA vaccine were cloned into a plasmid vector with backbone sequence elements (T7 promoter, 5′- and 3′-UTR, 100 nucleotide poly(A) tail) interrupted by a linker (A50LA50, 20 nucleotides) to improve RNA stability and translational efficiency. The DNA was purified, spectrophotometrically quantified, and in vitro-transcribed with an EZ™ T7 High Yield In Vitro Transcription kit (Enzynomics, Daejeon, South Korea) and a Cap 1 capping analog (SMARTCAP®, ST PHARM, Seoul, South Korea) and with N1-methylpseudouridine-5′-triphosphate (m1ΨTP; TriLink, CA, USA) to replace uridine-5′-triphosphate (UTP).
After transcription, RNA was purified by lithium chloride precipitation. dsRNA was eliminated by cellulose-based purification^{35 (link)}. RNA integrity was assessed using gel electrophoresis, and the concentration, pH, and endotoxin levels of the solution were determined.

Plasmid templates for the in vitro transcription of protein-encoding RNA were used as reporters. The Thy1.1 vector encodes the murine Thy1.1 protein, a highly conserved membrane glycoprotein. The Luc vector encodes for luciferase protein. Thy1.1 and Luc RNA were synthesized using 1-methyl-pseudouridine (N1-methylpseudouridine-5′-triphosphate, m1ΨTP, TriLink Biotechnologies, San Diego, CA, USA) [41 (link)] and double-stranded mRNA (dsmRNA) contaminants were removed via cellulose purification, as described [42 (link)]. The labeling of Luc RNA with fluorescent Cy5-UTP was performed according to the manufacturer’s instructions (BioNTech SE, Mainz, Germany). During the in vitro transcription of Luc RNA, 6% of total UTP was replaced with Cy5-labeled UTP.

RNA ranging in size from 100 nt to 9.4 kb was synthesized using the MEGAscript T7 transcription kit (Thermo Fisher Scientific, Invitrogen, Waltham, MA, USA). The reaction included UTP for generating standard IVT mRNA or N1-methylpseudouridine 5′-triphosphate (m1ΨTP) (TriLink) for nucleoside modified mRNA, in which 100% of uridine was substituted with m1Ψ. In a subset of RNAs, the sequences of the first three transcribed nucleotides were GCG, GGA, AGC, AGG or AGA. Cap analog, ARCA-G (TriLink, N-7003), beta-S-ARCA (D1, BioNTech SE) [17 (link)] or CleanCap^® Reagent AG (3′ OMe) (TriLink, N-7413) were added to the transcription reaction to generate mRNA with cap0 (A0), cap0 (D1) or cap1 (CC1), respectively. Vaccinia virus capping enzymes (New England Biolabs) were used according to the manufacturer’s instructions to enzymatically cap the synthesized mRNA and generate RNA with cap0 (E0) or cap1 (E1). RNA quality was tested using 1.4% agarose gel electrophoresis [18 (link)], and RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific).