1-MethylpseudoUridine - CAS 13860-38-3

N1-methyl-pseudoUridine (1-Methylpseudouridine), a methylpseudoUridine, outperforms 5 mC and 5 mC/N1-methyl-pseudoUridine in translation. N1-methyl-pseudoUridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density.

Catalog Number
Molecular Weight
Molecular Formula
N1-Methylpseudouridine; 2,4(1H,3H)-Pyrimidinedione, 1-methyl-5-b-D-ribofuranosyl-; (-)-1-Methyl-5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione; Antibiotic U-50228; U-50228; 1-N-Me-pseudouridine; (1S)-1,4-anhydro-1-(1-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-D-ribitol; Uracil, 1-methyl-5-b-D-ribofuranosyl-
Canonical SMILES
1.6±0.1 g/cm3
White to pale pink powder
Storage at 2-8°C


1-MethylpseudoUridine was first observed in the tRNAs of archaebacteria in which they were found to occur in the TΨC loop in place of ribothymidine. The base pairing properties and structure of this residue is very similar to uridine or ribothymidine. It may also confer an advantage in the thermal stability of the elbow region in tRNAs due to its enhanced stacking properties.

m1Ψ modification effect on protein expression

Chemically modified nucleotides play significant roles in the effectiveness of mRNA translation. Two sets of chemically modified mRNAs, which encoding firefly Luciferase (FLuc) and enhanced green fluorescent protein (eGFP), respectively, have been reported. They are used to evaluate the level of protein expression under different condition. The results indicate that chemical modifications of mRNAs are able to significantly improve protein expression, which is dependent on cell types and coding sequences. Among them, eGFP mRNAs with N1-methylpseudoUridine (m1ψ), 5-methoxyuridine, and pseudoUridine modifications all demonstrate a good performance to enhance protein expression.

m1Ψ modification improves mRNA switch performance

Synthetic mRNAs can be served as tools for controlling cell behavior owe to their modifiability and low genomic damage. Synthetic RNAs has multiple applications, such as biomolecule sensors, immune regulators, protein expression controllers and cell-fate controllers. Commonly, modified nucleosides, including pseudoUridine (Ψ) and 5-methyl-cytidine (m5C), are used to synthesize mRNA instead of uridine and cytidine, because they have little immune response to cells, to reduce the cytotoxic side effects of RNA transfection.

Studies in the effect of a series of naturally existing base modifications and their combinations on two synthetic mRNAs served as sensing RNA switch, including microRNA and RNA binding protein (RBP), have been reported. N1-methylpseudoUridine (m1Ψ) replacing uridine in synthetic mRNA avoids the cytotoxicity induced by immune response, and also substantially enhances the effectiveness of both types of switches. Compared with other modified bases, the m1Ψ substitution induced higher protein expression of mRNA, allowing clearer detection of signals and better fold-change between ON and OFF states of the switches. In addition, the m1Ψ substitution enhanced the sensitivity and performance of synthetic RNA composed of both miRNA- and RBP-sensing switches. The studies reveal m1Ψ as a prominent base substitution of uridine. Because m1Ψ has the potential for a wide range of applications in the design of synthetic RNAs that modulate various cell functions.

Low toxicity of m1Ψ modification in IVT RNA

RNA therapy is referred that in vitro-transcribed mRNA (IVT mRNA) is introduced to cells for therapeutic applications. IVT mRNA is transfected into target cells, translated, and the fully-synthesised encoded protein exerts a therapeutic effect via specific mechanisms. IVT mRNAs have been applied to treatment of inherited protein deficiencies, tissue reprogramming, cancer immunotherapy, genome editing, and vaccination against infectious diseases. However, using mRNAs for therapy has several problems. Cellular delivery of IVT mRNA activates the innate immune system via pattern recognition receptors leading to phosphorylation of eIF2α, global inhibition of translation, and upregulation of proinflammatory cytokines. Nucleotide modifications in IVT mRNA could partly resolve these problems by increasing the expression of the encoded protein and reducing innate immune activation compared to unmodified IVT mRNA.

Nevertheless, the modification IVT RNA in mRNA therapy may lead to off-target and long-term toxicity. There are some issues about immunogenicity of IVT mRNA and off-target effects, the effect of modified nucleotides degraded of modified IVT mRNA on mRNA translation and co-translational protein folding. Study have been investigated the off-target toxicity of three commonly modified nucleosides in IVT mRNA, including 5-methoxyuridine, 5-methylcytidine, and 1-methylpseudoUridine, which are cultured with human cells. Among them, 5-methoxyuridine exhibited cytotoxicity in A549 cells and HeLa cells at concentrations greater than 100 μM, while 1-methylpseudoUridine was nontoxic at all tested concentrations (up to 1000 μM), and 5-methylcytidine was more cytotoxic than other nucleosides (100 μM

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