In 2025, the cumulative number of mRNA vaccines administered globally has exceeded 10 billion doses, and its high efficiency and safety have completely rewritten the pattern of infectious disease prevention and control. From COVID-19 vaccines to cancer treatment, mRNA technology is penetrating the medical field at an alarming rate. However, back in 2005, the scientist Karikó's team first discovered that replacing uridine (U) in mRNA with pseudouridine (Ψ) can significantly reduce immunogenicity, and this discovery became the "Archimedes fulcrum" of mRNA therapy. But why can Ψ-RNA escape the security check of the immune system?
Toll-like receptors 7/8 (TLR7/8) in the immune system are like "nucleic acid detectors", which can specifically identify viral RNA degradation products and trigger inflammatory responses. However, the human body's own RNA can "get away with it" because it contains modifications such as Ψ. This phenomenon not only explains the low immunogenicity of mRNA vaccines, but also reveals the subtle mechanism of self-non-self recognition in the evolution of life.
Recently, in a research report titled "Pseudouridine RNA avoids immune detection through impaired endolysosomal processing and TLR engagement" published in the international journal Cell, scientists from the University of Munich and other institutions discovered for the first time that the "invisibility" of Ψ-RNA stems from the "selective blindness" of lysosomal nuclease RNase T2 and PLD exonuclease and the "double neglect" of TLR7/8 receptors.
The success of mRNA therapy relies on the immune silencing properties of Ψ modification, but its molecular mechanism has long been a mystery. In this study, the researchers aimed to reveal how Ψ-RNA achieves immune escape through the dual checkpoints of lysosomal processing and TLR recognition, thereby providing a certain theoretical basis for the design of safer RNA drugs.
In the article, the researchers used in vitro transcribed Ψ-RNA, m1Ψ-RNA (N1-methylpseudouridine) and unmodified RNA as models, and combined human monocytes, dendritic cells and TLR8/7 gene knockout cell lines to systematically evaluate differences in immune activation. The key reagents used in the study include RNase T2, PLD3/4 exonuclease and TLR-specific inhibitors (such as CU-CPT9a).
Figure 1. Ψ RNA does not activate TLR8. (Bérouti M, et al., 2025)
The study found that the cleavage efficiency of RNase T2 on Ψ-RNA was more than 90% lower than that of unmodified RNA, and PLD exonuclease also had difficulty degrading Ψ-RNA. In the cell model, Ψ-RNA could hardly activate TLR7/8-dependent inflammatory factors (such as IL-6, IFN-α), and although m1Ψ-RNA could weakly activate TLR8, it was still significantly weaker than unmodified RNA.
| Cat.No. | Product Name | Price |
|---|---|---|
| RSNK-016 | m7G(5')ppp(5')(2'OMeG)pG Cap Analog | Inquiry |
| RSNK-017 | m7(3'OMeG)(5')ppp(5')(2'OMeG)pG Cap Analog | Inquiry |
| RSNK-018 | Pseudo-UTP | Inquiry |
| RSNK-019 | 5-Methoxy-UTP | Inquiry |
| RSNK-020 | 5-Methyl-CTP | Inquiry |
| RSNK-023 | Pyrophosphatase, Inorganic | Inquiry |
| RSNK-024 | RNase Inhibitor, Murine | Inquiry |
| RSNK-025 | 2'-O-methyltransferase | Inquiry |
| RSNK-026 | Vaccinia Capping System | Inquiry |
| RSNK-027 | Poly (A) Polymerase | Inquiry |
| RSNK-028 | T7 High Yield RNA Synthesis Kit (Low dsRNA) | Inquiry |
| RSNK-029 | Thermostable T7 High Yield RNA Synthesis Kit | Inquiry |
| RSNK-030 | T7 RNA Polymerase (low dsRNA) | Inquiry |
| RSNK-031 | Thermostable T7 RNA Polymerase | Inquiry |
Further nuclear magnetic resonance (NMR) studies found that Ψ-RNA tends to form an A-type helical conformation, which will cause the B2 binding pocket of RNase T2 to be unable to adapt. At the same time, the methylation of N1 position of Ψ further hinders the binding of PLD exonuclease.
The immune silencing of Ψ-RNA is not a single mechanism, but a double insurance. On the one hand, RNase T2 and PLD exonuclease cannot effectively cut Ψ-RNA, resulting in the obstruction of TLR7/8 ligand generation. On the other hand, the first binding pocket of TLR8 ignores Ψ, and the second binding pocket of TLR7 rejects Ψ-RNA fragments.
In this study, researchers systematically revealed the molecular details of Ψ-RNA immune escape for the first time, which is of far greater significance than the field of mRNA vaccines. Ψ modification may be a self-marker evolved by eukaryotes, which can avoid autoimmune attacks by interfering with the recognition of nucleases and TLRs. This discovery not only consolidates the cornerstone of mRNA therapy, but also provides a new idea of "precisely regulating immunogenicity" for RNA drug design.
Although m1Ψ retains some TLR8 activation ability, its immunogenicity is still much lower than that of unmodified RNA, which suggests that in the future, RNA drugs may be able to balance efficacy and safety by fine-tuning the modification sites. The combination of LC-MS and NMR provides a high-resolution tool for RNA modification research, and in the future it may be possible to analyze the mechanisms of more immune stealth modifications (such as 2'-O-methylation).
Reference
Bérouti M, et al. Pseudouridine RNA avoids immune detection through impaired endolysosomal processing and TLR engagement. Cell, 2025.