Nucleobase-modified nucleotide for synthesis of mRNA
N1-Methylpseudouridine targets the translation machinery and the innate immune system. When incorporated into mRNA, it enhances translation by increasing ribosome density and promoting eIF2α-dependent and independent translation initiation. Additionally, N1-methylpseudouridine reduces the immunogenicity of mRNA by preventing recognition by Toll-like receptors (TLRs) such as TLR3, TLR7, and TLR8, which recognize unmodified RNA as foreign. This dual action—enhancing translation and reducing immune activation—makes N1-methylpseudouridine a critical component of modern mRNA therapeutics.

Nuclear modification of Luc and GFP mRNA with N1-methyl-pseudouridine improves the translation initiation step, partly through blocking eIF2α phosphorylation. In HEK293T cells, mRNA modified by inserting N1-methyl-pseudouridine produced the same amount of luc as standard Luc mRNA. Reduced formation elongation also led to increased polyribosomes and proliferation on Luc mRNA with NN1-methyl-pseudouridine. When Luc and GFP mRNA have access to N1-methyl-pseudouridine, translation is greatly improved in all external translation systems. Luc mRNA is not as crucial as N1-methyl-pseudouridine-Luc mRNA when it comes to polyribosomes [1].
N1-Methylpseudouridine exhibits in vitro activity by enhancing the translation efficiency of mRNA. In cell-free translation systems and cultured cells, mRNA containing N1-methylpseudouridine produces significantly more protein than mRNA containing unmodified uridine or other modified nucleosides such as 5-methylcytidine. The compound increases ribosome density on mRNA and promotes translation initiation. It also reduces the activation of innate immune sensors, leading to lower levels of type I interferon production. These in vitro activities have been extensively characterized in the context of mRNA vaccine development.

In mice and cell lines, N1-methylpseudouridine-incorporated mRNA combined with pseudouridine-incorporated mRNA can efficiently increase protein expression and decrease immunogenicity [2]. In vivo, m5C/N1-methyl-pseudouridine-modified mRNA is more potent than Ψ and m5C/Ψ-modified mRNA, as is the case with N1-methyl-pseudouridine (1-Mmethylpseudouridine) (20 μg; Im or id route 21 days). high proficiency in translation[2].
In vivo studies have demonstrated that N1-methylpseudouridine-modified mRNA produces higher protein expression and lower immunogenicity compared to unmodified mRNA. In animal models, mRNA vaccines containing N1-methylpseudouridine have shown superior efficacy in generating immune responses and protecting against viral challenge. The compound enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density. The reduced immunogenicity results in lower reactogenicity and better tolerability. These in vivo findings have been critical for the success of mRNA-based vaccines.

In vitro assays for N1-methylpseudouridine typically involve measuring translation efficiency of modified mRNA in cell-free or cell-based systems. Luciferase or GFP reporter mRNAs containing N1-methylpseudouridine are transcribed in vitro and transfected into cells. Protein expression is measured by luminescence or fluorescence. Ribosome density is measured by polysome profiling or ribosome footprinting. Immune activation is assessed by measuring type I interferon production (e.g., IFN-α, IFN-β) via ELISA or qPCR. These assays confirm the compound’s ability to enhance translation and reduce immunogenicity.

In vitro cellular assays for N1-methylpseudouridine are conducted in various cell types, including primary cells, dendritic cells, and cell lines such as HeLa or HEK293T. Cells are transfected with modified or unmodified mRNA encoding a reporter protein. Protein expression is measured over time by luminescence, fluorescence, or Western blot. Cell viability and cytotoxicity are assessed. Immune activation is measured by cytokine secretion (e.g., IFN-α, TNF-α) and by assessing activation of TLRs and other innate immune sensors. These experiments characterize the functional benefits of N1-methylpseudouridine modification.

Animal/Disease Models: 7weeks old balb/c (Bagg ALBino) mouse [1]
Doses: 20 μg
Route of Administration: intramuscularor injection route, lasting for 21 days
Experimental Results: It has high translation ability.
In vivo animal experiments with N1-methylpseudouridine-modified mRNA are conducted in mouse models. mRNA encoding a reporter protein (e.g., luciferase) or an antigen (e.g., SARS-CoV-2 spike protein) is administered via intramuscular or intravenous injection. Protein expression is measured by in vivo imaging (for luciferase) or by ELISA for antigen-specific antibodies. Immune responses are assessed by measuring antibody titers and T cell responses. Reactogenicity is assessed by measuring weight loss, fever, and cytokine levels. These studies have been critical for the development of mRNA vaccines.

Pharmacokinetic data for N1-methylpseudouridine are relevant in the context of mRNA therapeutics. When incorporated into mRNA, the modified nucleoside is delivered to cells as part of the mRNA molecule. The mRNA is translated in the cytoplasm, and the protein product is secreted or retained intracellularly. The modified nucleoside itself is not a free drug but a component of the mRNA. The half-life of mRNA in cells is influenced by the modification, with N1-methylpseudouridine contributing to increased mRNA stability and translation efficiency. The compound is metabolized as part of the mRNA degradation pathway.

N1-Methylpseudouridine has been extensively evaluated for safety in the context of mRNA vaccines. It has been shown to be well-tolerated in preclinical and clinical studies. The compound does not cause significant cytotoxicity or genotoxicity. Its reduced immunogenicity compared to unmodified RNA results in lower reactogenicity. The safety profile of N1-methylpseudouridine has been demonstrated in billions of doses of mRNA vaccines administered globally. It is considered a safe and effective component of mRNA therapeutics.

[1]. N1-methyl-pseudouridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density. Nucleic Acids Res. 2017 Jun 2;45(10):6023-6036.

[2]. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release. 2015 Nov 10;217:337-44.

[3]. Nance KD, Meier JL. Modifications in an Emergency: The Role of N1-Methylpseudouridine in COVID-19 Vaccines. ACS Cent Sci. 2021;7(5):748-756.

1-Methylpseuuridine is a methylpseuuridine in which the methyl group is located at the N-1 position of the uracil ring. 1-Methylpseuuridine has been reported in Streptomyces platensis and Streptomyces lincolnensis, with relevant data available. Certain chemical modifications can enhance the stability of in vitro transcribed mRNA and reduce its immunogenicity, thereby promoting the expression of proteins with therapeutic value. This paper demonstrates that N1-methylpseuuridine (N1mΨ) is superior to several other nucleoside modifications and their combinations in terms of translational ability. Through extensive analysis of various modified transcripts in a cell-free translation system, we elucidated its effects on protein expression and ruled out the influence of mRNA stability mechanisms. We found that, in addition to shutting down immune/eIF2α phosphorylation-dependent translational repression, incorporated N1mΨ nucleotides significantly alter the dynamics of the translation process by increasing the ribosome pauses and density on mRNA. Our results suggest that the increased ribosome loading on modified mRNA makes it easier to initiate translation, which can be achieved by promoting ribosome cycling on the same mRNA or de novo recruitment of ribosomes. [1] Messenger RNA is gaining popularity as a therapeutic tool in the field of gene therapy. It has been recognized that nucleobase modifications can greatly enhance the properties of mRNA by reducing immunogenicity and increasing the stability of RNA molecules (Kariko paradigm), which is crucial for this revolution. We found that N(1)-methylpseudouridine (m1Ψ) mRNA modified alone or in combination with 5-methylcytidine (m5C) showed reporter gene expression levels approximately 44-fold (dual-modified mRNA group) and approximately 13-fold (single-modified mRNA group) higher, respectively, than state-of-the-art pseudouridine (Ψ) and/or m5C/Ψ modified mRNA platforms after transfection of cell lines or mice. We also found that (m5C/)m1Ψ modified mRNA reduced intrinsic intracellular immunogenicity and increased cell viability after in vitro transfection compared to (m5C/)Ψ modified mRNA. The enhanced protein expression capacity of (m5C/)m1Ψ-modified mRNAs may be at least partly attributed to their enhanced ability to evade endosomal Toll-like receptor 3 (TLR3) activation and downstream innate immune signaling. We believe that the (m5C/)m1Ψ-mRNA platform proposed in this paper has the potential to become a new standard in the field of modified mRNA therapies. [2] The novel coronavirus SARS-CoV-2, the pathogen of the COVID-19 pandemic, has spurred one of the most efficient vaccine development campaigns in human history. A key aspect of COVID-19 mRNA vaccines is the use of modified nucleobase N1-methylpseudouridine (m1Ψ) to enhance their effectiveness. In this outlook, we summarize the development and function of m1Ψ in synthetic mRNAs. By elucidating the mechanism of action of this novel element in these drugs, we aim to enhance understanding and highlight opportunities for future chemical innovation. [3]
N1-Methylpseudouridine is a key component of modern mRNA therapeutics, including COVID-19 vaccines. It enhances translation efficiency by increasing ribosome density and promotes eIF2α-dependent and independent translation initiation. The compound also reduces immunogenicity by preventing recognition by TLRs. It is also known as 1-methylpseudouridine or m1Ψ. Its use has revolutionized mRNA-based drug development. The compound is available in high purity for research and manufacturing applications.

C₁₀H₁₄N₂O₆

258.23

258.085

C, 46.51; H, 5.46; N, 10.85; O, 37.17

13860-38-3

N1-Methylpseudouridine-5′-triphosphate trisodium;N1-Methylpseudouridine-5′-triphosphate;1428903-59-6;N1-Methylpseudouridine-d3

99543

White to off-white solid powder

1.576g/cm3

189 °C

1.618

-2.6

4

6

2

18

409

4

CN1C=C(C(=O)NC1=O)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O

UVBYMVOUBXYSFV-XUTVFYLZSA-N

InChI=1S/C10H14N2O6/c1-12-2-4(9(16)11-10(12)17)8-7(15)6(14)5(3-13)18-8/h2,5-8,13-15H,3H2,1H3,(H,11,16,17)/t5-,6-,7-,8+/m1/s1

5-[(2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1-methylpyrimidine-2,4-dione

N1Methylpseudouridine; 1-Methylpseudouridine; 13860-38-3; N1-Methylpseudouridine; N1-methyl-pseudouridine; m(1)f; 09RAD4M6WF; 5-((2S,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1-methylpyrimidine-2,4(1H,3H)-dione; 2,4(1H,3H)-Pyrimidinedione, 1-methyl-5-beta-D-ribofuranosyl-; N1 Methylpseudouridine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~125 mg/mL (~484.1 mM)
H2O: ~50 mg/mL (~193.6 mM

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (193.63 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)