Modifications in an Emergency: The Role of N1-Methylpseudouridine in COVID-19 Vaccines

Abstract

The novel coronavirus SARS-CoV-2, the cause of the COVID-19 pandemic, has inspired one of the most efficient vaccine development campaigns in human history. A key aspect of COVID-19 mRNA vaccines is the use of the modified nucleobase N1-methylpseudouridine (m1Ψ) to increase their effectiveness. In this Outlook, we summarize the development and function of m1Ψ in synthetic mRNAs. By demystifying how a novel element within these medicines works, we aim to foster understanding and highlight future opportunities for chemical innovation.

Figure 1

(a) mRNA-based COVID-19 vaccine strategy. (b) Structural features of uridine and m1Ψ. TCR = T-cell receptor. MHC = major histocompatibility complex.

Figure 2

Top: Design elements found in synthetic mRNA therapeutics. Bottom: Sequence of the COVID-19 mRNA vaccine tozinameran (BNT162b2) from Pfizer/BioNTech. Green: 5′-cap. Yellow: 5′- and 3′-UTR sequences. Blue: SARS-CoV-2 spike glycoprotein coding sequence. Red: Segmented poly(A) tail.

Figure 3

Production of m1Ψ mRNAs by in vitro transcription. Left: Components of in vitro transcription reaction. Right: Incorporation of m1Ψ-triphosphate into RNA is guided by m1Ψ’s ability to form a canonical base pair with adenine of the DNA template in the T7 RNA polymerase active site.

Figure 4

(a) Activation of innate immune response by mRNA secondary structures (b) Structure of the single-stranded RNA sensor TLR7 in complex with a polyuridine (poly(U)) ligand (PDB ID: 5GMF). Replacing uridine with m1Ψ demonstrates the steric incompatibility of the modified nucleobase with TLR7 binding and immune activation.

Figure 5

m1Ψ exerts context-dependent effects on translation. Left: m1Ψ-dependent enforcement of secondary structure in the 5′-UTR of synthetic mRNAs can inhibit translation initiation. Right: m1Ψ-dependent enforcement of secondary structure in the coding sequences of synthetic mRNAs can increase their functional half-life. Note: While m1Ψ is homogeneously incorporated throughout synthetic mRNA vaccines, in these illustrations, m1Ψ is only specified in duplexes to emphasize its potential to influence mRNA structure.