Pseudouridine-Modified mRNA: How It Evades Immune Detection via TLR7/8 Suppression

September 15, 2025
 |  No Comments

Introduction

Toll-like receptors (TLRs) play a crucial role in innate immunity by detecting foreign RNA and triggering immune responses. Among these, TLR7 and TLR8 recognize RNA degradation products in endolysosomal compartments, activating antiviral defenses. However, endogenous RNA modifications, such as pseudouridine (Ψ), enable RNA to evade immune detection—a feature critical for the success of mRNA-based therapeutics.

Despite the widespread use of Ψ-modified mRNA in vaccines and therapies, the molecular mechanisms underlying its immune evasion properties have remained unclear. In a groundbreaking study published in Cell, Bérouti et al. systematically dissect how Ψ-modified RNA avoids TLR7/8 activation by impairing endolysosomal processing and reducing receptor engagement. Their findings also reveal an unexpected TLR8-stimulatory effect of N1-methylpseudouridine (m1Ψ), a common therapeutic modification, raising important considerations for future mRNA drug design.

Key Findings

1. Pseudouridine-Modified RNA Resists Endolysosomal Degradation

TLR7/8 activation depends on the cleavage of RNA by endolysosomal nucleases, particularly RNase T2, which preferentially cuts at purine-uridine (RU) dinucleotide sites. The study demonstrates that Ψ modification disrupts RNase T2 activity, preventing the generation of immunostimulatory RNA fragments.

  • Mass spectrometry analysis confirmed that Ψ-modified RNA is poorly processed by RNase T2.
  • Exonucleases PLD3/PLD4, which further degrade RNA fragments for TLR activation, also exhibit reduced activity against Ψ-RNA.
  • Structural studies revealed that Ψ stabilizes RNA in an A-helical conformation, hindering nuclease binding and cleavage.

As a result, Ψ-modified in vitro transcribed (IVT) RNA failed to induce TLR7/8-dependent cytokine secretion in human primary monocytes, plasmacytoid dendritic cells (pDCs), and BLaER1 monocyte models.

RNase T2 or RNase 1 cleavage patterns of Ψ-modified vs. unmodified RNA

Figure 1 RNase T2 or RNase 1 cleavage patterns of Ψ-modified vs. unmodified RNA1,2

2. Weak TLR8 Activation by Pseudouridine

Beyond impaired RNA processing, Ψ itself is a poor activator of TLR8.

  • Biochemical assays showed that while uridine (U) and m1Ψ induce TLR8 dimerization (a hallmark of receptor activation), Ψ has minimal activity.
  • When co-delivered with RNA fragments occupying TLR8’s second binding pocket, Ψ exhibited weak synergistic activation, suggesting it retains some binding capacity.

Notably, m1Ψ—a common mRNA therapeutic modification—retained TLR8-stimulatory activity similar to U, despite resisting nuclease degradation. This finding has implications for mRNA vaccine design, as m1Ψ may enhance translation efficiency but also carry a risk of off-target immune activation.

2 Ψ is a weak agonist of TLR8 compared to U and m1Ψ

Figure 2 Ψ is a weak agonist of TLR8 compared to U and m1Ψ3,2

3. Dual Mechanisms of TLR7 Evasion

TLR7 activation requires two ligands:

  • 2′,3′-cyclic guanosine monophosphate (2′,3′-cGMP) (binding pocket 1)
  • Short oligonucleotides (ORN) (binding pocket 2)

Ψ-modified RNA disrupts this process in two ways:

  • Impaired generation of 2′,3′-cGMP due to resistance to RNase T2 and PLD nucleases.
  • Failure of Ψ-containing ORNs to activate TLR7, even when 2′,3′-cGMP is provided exogenously.

Mouse studies confirmed that RNase T2 knockout significantly reduced IFNα responses to unmodified mRNA, highlighting its essential role in TLR7-dependent RNA sensing.

3 Ψ inhibits 2′,3′-cGMP release required for TLR7 activation

Figure 3 Ψ inhibits 2′,3′-cGMP release required for TLR7 activation4,2

Implications for mRNA Therapeutics and Immune Tolerance

This study presents an integrated model for how Ψ-modified RNA evades TLR7/8 detection:

  • Obstructs nuclease processing (RNase T2/PLD3/PLD4)
  • Reduces TLR8 binding affinity
  • Prevents TLR7 ligand generation and engagement

Key Takeaways:

  • Ψ-modified mRNA avoids immune detection, explaining its success in vaccines.
  • m1Ψ retains TLR8-stimulatory activity, potentially leading to unintended immune responses.
  • Endogenous Ψ modifications may help distinguish self vs. non-self RNA, preventing autoimmunity.

Future research should explore whether other RNA modifications (e.g., 2′-O-methylation) cooperate with Ψ to maintain immune tolerance.

Applications & Product Recommendation

If you are developing mRNA-based therapeutics, vaccines, or gene therapies, selecting the right nucleoside modifications is critical for balancing translation efficiency and immune safety.

Our Recommended Solution:

BOC Sciences’s High-Purity Pseudouridine Nucleoside

  • ≥99% purity, GMP-grade or research-grade available
  • Optimized for in vitro transcription (IVT) and large-scale mRNA synthesis
  • Enhances RNA stability and reduces innate immune activation
  • Compatible with lipid nanoparticle (LNP) delivery systems

Why choose our pseudouridine?

  • Proven to reduce TLR7/8 recognition
  • Supports higher mRNA translation without triggering strong cytokine responses
  • Trusted by vaccine manufacturers and biotech R&D teams

Contact us today to request a free sample or bulk pricing.

Commom PseudoUridine at BOC Sciences

CASProduct NameCategory
1445-07-4β-pseudoUridineUnmodified pseudoUridine
39967-60-72′-DeoxypseudoUridineUnmodified pseudoUridine
10017-66-0α-pseudoUridineUnmodified pseudoUridine
64272-68-01,3-DimethylpseudoUridineBase modified pseudoUridine
81691-06-7N3-MethylpseudoUridineBase modified pseudoUridine
13860-38-3N1-MethylpseudoUridineBase modified pseudoUridine
1157-60-4PseudoUridine 5′-monophosphateMonophosphate pseudoUridine
 PseudoUridine-5′-TriphosphateTriphosphate pseudoUridine
 N1-MethylpseudoUridine-5′-Triphosphate SodiumTriphosphate pseudoUridine
 PseudoUridine 5′-Triphosphate SodiumTriphosphate pseudoUridine

Conclusion

The work by Bérouti et al. provides a mechanistic foundation for understanding how Ψ-modified RNA evades TLR7/8 detection, offering critical insights for mRNA vaccine and therapeutic design. Their discovery that m1Ψ retains TLR8 activity suggests a trade-off between translation efficiency and immunogenicity, guiding future optimization of synthetic mRNA constructs.

For more details, read the full paper:

Bérouti M, Wagner M, Greulich W, et al. Pseudouridine RNA avoids immune detection through impaired endolysosomal processing and TLR engagement. Cell. 2025. doi: 10.1016/j.cell.2025.05.032

References

  1. Image retrieved from Figure 1 “Characterization of ssRNA digested with RNase T2 or RNase 1,” Bérouti, Marleen, et al., 2025, used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was modified by extracting and using only part a, and the title was changed to “RNase T2 or RNase 1 cleavage patterns of Ψ-modified vs. unmodified RNA”
  2. Bérouti, Marleen, et al. “Pseudouridine RNA avoids immune detection through impaired endolysosomal processing and TLR engagement.” Cell (2025).
  3. Image retrieved from Figure 5 “Ψ is a poor ligand for TLR8,” Bérouti, Marleen, et al., 2025, used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was modified by extracting and using only part a, and the title was changed to “Ψ is a weak agonist of TLR8 compared to U and m1Ψ”
  4. Image retrieved from Figure 6 “Ψ inhibits the release of 2′,3′-cGMP for TLR7 activation,” Bérouti, Marleen, et al., 2025, used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was modified by extracting and using only part a, and the title was changed to “Ψ inhibits 2′,3′-cGMP release required for TLR7 activation”
Uncategorized