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

1. N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes
Kei Araki, Hirofumi Watanabe, Hiroki Kohno, Eiji Sugiyama, Sho Mokuda, Michinori Ishitoku, Shintaro Hirata Cytotechnology . 2022 Aug;74(4):503-514. doi: 10.1007/s10616-022-00540-4.
Studies conducted using murine arthritis models have indicated that the use of in vitro-transcribed messenger RNA (IVT mRNA) is an effective therapeutic approach for joint diseases. However, the use of IVT mRNA in human synovial cells has not been widely studied. Recently, the outbreak of the novel coronavirus disease has accelerated the development of innovative mRNA vaccines, such as those containing a modified nucleic acid, N1-methylpseudouridine-5'-triphosphate (m1ψ). IVT mRNA is an attractive tool for biological experiments and drug discovery. To verify the protein expression from IVT mRNA in vitro, primary cultured fibroblast-like synoviocytes (FLS) and MH7A human synovial fibroblast cells were transfected with enhanced green fluorescent protein (EGFP) mRNA with or without m1ψ incorporation. EGFP was detected using western blotting and fluorescence microscopy. A multiplex assay was performed to comprehensively understand IVT mRNA-induced immunogenicity. Gene expression levels were measured using reverse transcription polymerase chain reaction. In both MH7A cells and FLS, cells transfected with EGFP mRNA containing m1ψ generated higher levels of EGFP than those transfected with unmodified EGFP or control mRNAs. The multiplex assay of the FLS culture supernatant and reverse transcription polymerase chain reaction for FLS revealed that both concentration and expression of IL-6, TNF-α, and CXCL10 were upregulated by unmodified EGFP mRNA, whereas they were suppressed by EGFP mRNA with m1ψ. Overall, m1ψ incorporation enhanced protein expression and decreased the expression of cytokines. These findings may contribute to arthritis research.
2. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes
Michael J Hope, Barbara L Mui, Hiromi Muramatsu, Steven Tuyishime, Norbert Pardi, Ying K Tam, Drew Weissman, Katalin Kariko, Thomas D Madden J Control Release . 2015 Nov 10;217:345-51. doi: 10.1016/j.jconrel.2015.08.007.
In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005-0.250mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1-4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins.
3. Human 5' UTR design and variant effect prediction from a massively parallel translation assay
Georg Seelig, David W Reid, Iain J McFadyen, Ban Wang, Paul J Sample, Vlad Presnyak, David R Morris Nat Biotechnol . 2019 Jul;37(7):803-809. doi: 10.1038/s41587-019-0164-5.
The ability to predict the impact of cis-regulatory sequences on gene expression would facilitate discovery in fundamental and applied biology. Here we combine polysome profiling of a library of 280,000 randomized 5' untranslated regions (UTRs) with deep learning to build a predictive model that relates human 5' UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5' UTRs that accurately direct specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,212 truncated human 5' UTRs and 3,577 naturally occurring variants and show that the model predicts ribosome loading of these sequences. Finally, we provide evidence of 45 single-nucleotide variants (SNVs) associated with human diseases that substantially change ribosome loading and thus may represent a molecular basis for disease.

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