evidence: Whereas both Pfizer-BioNTech and Moderna COVID-19 vaccines encode the identical spike protein of SARS-CoV-2, their nucleotide sequences, especially at the wobble position, are substantially different due to the different codon optimization strategies used (Fig. 6a). Moderna has replaced most of the adenine and thymine at wobble position with guanine and cytosine, which leads to 14.5% higher GC content at the wobble position of the spike gene than that of Pfizer-BioNTech. Although high GC content might be beneficial for mRNA translation46,47 or stabilization48, it reduces the content of uridine in mRNA that can be replaced by m1Ψ during in vitro transcription. M1Ψ enhances vaccine efficacy by stabilizing mRNA and facilitating evasion from host innate immunity30,31. To explore whether m1Ψ content, especially that at the wobble position (wobble m1Ψ), influences vaccine efficacy, three mRNAs encoding the identical Receptor Binding Domain (RBD) of SARS-CoV-2 spike but containing varying levels of wobble m1Ψ were generated (Fig. 6b). Briefly, RBD-V5 mRNA contains the highest wobble m1Ψ (29.7%), while RBD-V6 (10.8%) and -V7 (0%) mRNAs contain wobble m1Ψ content close to that of Pfizer-BioNTech (10.9%) and Moderna (2.6%) spike mRNA, respectively.