Nucleobase-modified nucleotide for synthesis of mRNA

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].

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].

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.

[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-methylpseudouridine is a methylpseudouridine in which the methyl group is located at position N-1 on the uracil ring.
1-Methylpseudouridine has been reported in Streptomyces platensis and Streptomyces lincolnensis with data available.

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Certain chemical modifications confer increased stability and low immunogenicity to in vitro transcribed mRNAs, thereby facilitating expression of therapeutically important proteins. Here, we demonstrate that N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. Through extensive analysis of various modified transcripts in cell-free translation systems, we deconvolute the different components of the effect on protein expression independent of mRNA stability mechanisms. We show that in addition to turning off the immune/eIF2α phosphorylation-dependent inhibition of translation, the incorporated N1mΨ nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Our results indicate that the increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo ribosome recruitment.[1]

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Messenger RNA as a therapeutic modality is becoming increasingly popular in the field of gene therapy. The realization that nucleobase modifications can greatly enhance the properties of mRNA by reducing the immunogenicity and increasing the stability of the RNA molecule (the Kariko paradigm) has been pivotal for this revolution. Here we find that mRNAs containing the N(1)-methylpseudouridine (m1Ψ) modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the current state-of-the-art pseudouridine (Ψ) and/or m5C/Ψ-modified mRNA platform by providing up to ~44-fold (when comparing double modified mRNAs) or ~13-fold (when comparing single modified mRNAs) higher reporter gene expression upon transfection into cell lines or mice, respectively. We show that (m5C/)m1Ψ-modified mRNA resulted in reduced intracellular innate immunogenicity and improved cellular viability compared to (m5C/)Ψ-modified mRNA upon in vitro transfection. The enhanced capability of (m5C/)m1Ψ-modified mRNA to express proteins may at least partially be due to the increased ability of the mRNA to evade activation of endosomal Toll-like receptor 3 (TLR3) and downstream innate immune signaling. We believe that the (m5C/)m1Ψ-mRNA platform presented here may serve as a new standard in the field of modified mRNA-based therapeutics.[2]

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The novel coronavirus SARS-CoV-2, the cause of the COVID-19 pandemic, has inspired one of the most efficient vaccine development campaigns in human history. A key aspect of COVID-19 mRNA vaccines is the use of the modified nucleobase N1-methylpseudouridine (m1Ψ) to increase their effectiveness. In this Outlook, we summarize the development and function of m1Ψ in synthetic mRNAs. By demystifying how a novel element within these medicines works, we aim to foster understanding and highlight future opportunities for chemical innovation.[3]

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.)