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-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]

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.)