Nucleobase-modified nucleotide for synthesis of mRNA

用 N1-甲基-假尿苷对 Luc 和 GFP mRNA 进行核修饰可改善翻译起始步骤，部分是通过阻断 eIF2α 磷酸化。在 HEK293T 细胞中，通过插入 N1-甲基-假尿苷修饰的 mRNA 产生与标准 Luc mRNA 相同量的 luc。形成伸长的减少还导致多核糖体增加以及带有 NN1-甲基-假尿苷的 Luc mRNA 的增殖。当 Luc 和 GFP mRNA 能够接触到 N1-甲基-假尿苷时，所有外部翻译系统中的翻译都会得到极大改善。对于多核糖体而言，Luc mRNA 并不像 N1-甲基-假尿苷-Luc mRNA 那样重要[1]。

在小鼠和细胞系中，N1-甲基假尿苷掺入的 mRNA 与假尿苷掺入的 mRNA 结合可以有效增加蛋白表达并降低免疫原性 [2]。在体内，m5C/N1-甲基-假尿苷修饰的 mRNA 比 Ψ 和 m5C/Ψ 修饰的 mRNA 更有效，就像 N1-甲基-假尿苷 (1-M甲基假尿苷) (20 μg；Im 或 id 途径 21天）。翻译水平较高[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

124.78

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

配方 1 中的溶解度: ≥ 2.08 mg/mL (8.05 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (8.05 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (8.05 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 50 mg/mL (193.63 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。