Nucleobase-modified nucleotide for synthesis of mRNA
N1-Methylpseudouridine-5′-triphosphate targets RNA polymerases, serving as a substrate for incorporation into mRNA during in vitro transcription using T7 RNA polymerase. Once incorporated into mRNA, N1-methylpseudouridine replaces uridine and modifies the molecular properties of the RNA. This modification reduces the activation of Toll-like receptors (TLRs) and other innate immune sensors, thereby decreasing the immunogenicity of the mRNA. The compound also enhances mRNA stability and translational efficiency, improving protein expression from the mRNA. Its ability to modulate the immune recognition of mRNA makes it a valuable tool for developing mRNA-based therapeutics and vaccines.

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 researchers 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. researchers 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. It is believed that the (m5C/)m1Ψ-mRNA platform presented here may serve as a new standard in the field of modified mRNA-based therapeutics[1].

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In vitro, N1-Methylpseudouridine-5′-triphosphate is used as a substrate for in vitro transcription reactions catalyzed by T7 RNA polymerase. The modified nucleotide is incorporated into mRNA transcripts, replacing uridine. The resulting modified mRNA shows reduced immunogenicity in cell-based assays, as measured by decreased activation of TLRs and reduced production of pro-inflammatory cytokines such as type I interferons. Modified mRNA also shows enhanced stability and translational efficiency compared to unmodified mRNA, leading to higher protein expression levels. The compound's activity is typically assessed by measuring reporter gene expression, cytokine production, and cell viability in cells transfected with modified mRNA. Detailed quality control data are available from commercial suppliers.

In vivo, N1-Methylpseudouridine-5′-triphosphate is used in the production of mRNA-based vaccines and therapeutics. mRNA containing this modified nucleotide has been shown to elicit robust immune responses with reduced adverse effects in animal models and in humans. The modification reduces the immunogenicity of the mRNA, allowing for higher and more durable protein expression. This has been critical for the development of COVID-19 mRNA vaccines, which incorporate N1-methylpseudouridine to improve efficacy and safety. The compound's in vivo efficacy is typically assessed by measuring antigen-specific antibody responses, T-cell responses, and protection against infectious agents in animal models. The compound is not administered directly but is used in the manufacturing of mRNA therapeutics.

In vitro mRNA lipofection and firefly luciferase assay[1]
mRNA was mixed with Lipo 2 K at a ratio of 1:2 (μg mRNA: μl Lipo 2 K) in Opti-MEM I. The complexes were allowed to form for 30 min at room temperature. The average hydrodynamic size and zeta potential of the RNA/lipid complexes were determined as described previously by dynamic light scattering and laser Doppler electrophoresis using a Zetasizer Nano ZS. Transfections were performed by adding 1 μg of complexed mRNA to cells pre-seeded in 24 well plates. The complexes were removed 4 h later along with Opti-MEM I and were replaced with the ATCC recommended culture media containing serum. 24 h after transfection, cells were lysed with 100 μl of 1 × Passive Lysis Buffer and firefly luciferase activity was measured with the Luciferase Assay Kit according to the manufacturer's protocol. Bioluminescence was measured using the GloMax luminometer.

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Enzyme-linked immunosorbent assay (ELISA)[1]
Cell culture supernatants were collected 24 h after transfection with mRNA and stored at − 80 °C until the ELISAs were performed unless stated otherwise. The ELISA kits for human interferon-β (IFN-β) and chemokine (C–C motif) ligand 5 (CCL5; also known as RANTES) were purchased from BioLegend and Life Technologies, respecitively. ELISAs were performed according to the manufacturers' recommendations, as described previously.

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Protein concentration assay[1]
Protein concentrations of cell lysates were measured using the Pierce™ Micro BCA™ Protein Assay kit. The standard manufacturer's protocol was followed after lysing the cells with Passive Lysis Buffer and diluting the lysate (1:10) in water.

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The in vitro transcription assay for N1-Methylpseudouridine-5′-triphosphate typically uses a template DNA encoding a reporter gene or antigen, T7 RNA polymerase, and a mixture of NTPs including ATP, GTP, CTP, and the modified UTP. The reaction is performed in a suitable buffer at 37°C for 2-4 hours. The resulting mRNA is purified and analyzed for integrity and yield by agarose gel electrophoresis or capillary electrophoresis. The incorporation efficiency of the modified nucleotide is assessed by mass spectrometry or enzymatic digestion followed by HPLC analysis. For functional assays, the modified mRNA is transfected into cells using lipid-based transfection reagents, and protein expression is measured by Western blotting, ELISA, or flow cytometry. Immunogenicity is assessed by measuring cytokine production or TLR activation in reporter cell lines.

Viability assayv[1]
Mammalian cells were transfected in 24 well plates with 1 μg of unmodified or modified RNA as described above. 24 h after transfection, the viability of mRNA-transfected cells was measured using an MTT proliferation assay according to the manufacturer's protocol.

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Immunostaining of TLR3 and flow cytometry[1]
24 h after transfection with mRNA, cells were collected, washed with phosphate buffered saline without calcium or magnesium (DPBS, no calcium, no magnesium) and incubated at room temperature for 1 h in 1 x Fixation Buffer. The fixed cells were washed twice with 1 × Permeabilization Buffer, resuspended in 1 × Permeabilization Buffer and incubated in the dark for 30 min with Phycoerythrin (PE)-conjugated anti-TLR3 antibodies. The cells were then washed twice with PBS to get rid of any unbound antibodies, and resuspended in PBS. Fluorescence signal was measured by flow cytometry on a BD Accuri™ C6 flow cytometer. Data were analyzed using the CFlow Plus Analysis software. Live cells were gated based on the areas of the forward/side scatters and single cells were gated based on the width of the forward scatter. The mean fluorescence intensity (MFI) of the PE dye (excitation laser: 488 nm, emission filter: 585/40 nm) was calculated based on the live, single cell population.

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For in vitro cellular assays, cells such as HEK293T, HeLa, or dendritic cells are transfected with mRNA containing N1-Methylpseudouridine-5′-triphosphate at varying concentrations (typically 0.1 to 10 µg/mL). Protein expression is assessed by Western blotting, ELISA, or flow cytometry at 6-48 hours post-transfection. Immunogenicity is assessed by measuring type I interferon production (IFN-α, IFN-β) and pro-inflammatory cytokines (TNF-α, IL-6) by ELISA or qRT-PCR. TLR activation is measured using TLR reporter cell lines. Cell viability is assessed using MTT or CellTiter-Glo assays. For comparison, cells are also transfected with unmodified mRNA containing uridine. All experiments include appropriate controls (unmodified mRNA, vehicle) and are performed in triplicate.

Mouse experiments[1]
7-week-old Balb/c mice were housed in individually ventilated cages under 12:12 h dark–light cycle conditions. Access to food and water was maintained ad libitum. Mice were anesthetized with constant flow of isoflurane during intradermal (i.d.) or intramuscular (i.m.) injections. 20 μg of mRNA were complexed with Lipo 2 K at a 1:1 (μg mRNA: μl Lipo 2 K) ratio and resuspended in PBS and injected i.d. or i.m into the tibialis anterior muscle.

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In vivo imaging of firefly luciferase expression[1]
The expression levels of firefly luciferase in mice were measured over time using the in vivo bioluminescent imaging system, IVIS Lumina II, until the signal from the injected mRNA reached background levels. Mice were injected intraperitoneally (i.p.) with 50 mg/kg of D-luciferin and bioluminescence was measured 10 min after the injection. Acquisition settings were set at f-stop: 1, binning: 8, and auto-exposure.

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For in vivo efficacy studies, animal models are used to evaluate the immunogenicity and protective efficacy of mRNA vaccines containing N1-Methylpseudouridine-5′-triphosphate. Mice or non-human primates are immunized with mRNA-LNP (lipid nanoparticle) formulations encoding antigens such as SARS-CoV-2 spike protein or influenza hemagglutinin. The mRNA is administered via intramuscular or intradermal injection at doses ranging from 1 to 100 µg per animal. Antigen-specific antibody responses are measured by ELISA, and neutralizing antibody titers are assessed by plaque reduction neutralization tests or pseudovirus neutralization assays. T-cell responses are assessed by ELISpot or intracellular cytokine staining. Protection against challenge with infectious agents is assessed by measuring viral loads and survival. All animal procedures are conducted in accordance with institutional guidelines.

The pharmacokinetic properties of N1-Methylpseudouridine-5′-triphosphate are not typically evaluated as a direct therapeutic agent, as it is a nucleotide substrate used in mRNA manufacturing. Once incorporated into mRNA and formulated into lipid nanoparticles, the pharmacokinetics of the mRNA-LNP formulation are determined by the nanoparticle properties rather than the nucleotide itself. The mRNA is distributed to tissues including liver, spleen, and lymph nodes following intramuscular or intravenous administration. The mRNA is translated into protein in the cytoplasm and is eventually degraded by cellular nucleases. The modified nucleotide itself does not persist in the body as the mRNA is cleared over time. The compound is for research use only and is not administered directly to patients.

The toxicology of N1-Methylpseudouridine-5′-triphosphate is primarily evaluated in the context of mRNA-LNP formulations. The modified nucleotide itself is considered to have low toxicity, as it is a naturally occurring modified nucleoside. In preclinical toxicology studies of mRNA-LNP formulations containing N1-methylpseudouridine, the formulations show a favorable safety profile with no significant adverse effects at therapeutic doses. Local injection site reactions and transient inflammatory responses may occur, consistent with the pharmacological activity of the mRNA vaccine. The compound is not genotoxic or carcinogenic. The safety profile of N1-methylpseudouridine-containing mRNA has been extensively evaluated in clinical trials for COVID-19 vaccines and supports its use in mRNA therapeutics.

[1]. Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T. 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;217:337-344.

N1-Methylpseudouridine-5′-triphosphate is a modified nucleotide used in the synthesis of mRNA for therapeutics and vaccine development. Incorporation of this nucleotide reduces mRNA immunogenicity and enhances stability and translational efficiency. It is a critical component of mRNA-based COVID-19 vaccines. The compound is not approved as a therapeutic agent itself but is used as a raw material in the manufacturing of mRNA therapeutics. It is available as a high-purity research reagent (≥99%) for research and manufacturing use.

C10H17N2O15P3

498.1677

497.984

1428903-59-6

N1-Methylpseudouridine;13860-38-3;N1-Methylpseudouridine-5′-triphosphate trisodium;N1-Methylpseudouridine-5′-triphosphate tetralithium

90456815

RNA1{[*P(=O)(O)OP(=O)(O)OP(=O)(O)O |$_R2;;;;;;;;;;;;|]R([*c1cn(c(=O)[nH]c1=O)C |$_R1;;;;;;;;;$|])}$$$$

P-P-P-m1Yra-Ribf

Colorless to light yellow liquid

2.010±0.06 g/cm3

-6.5

7

15

8

30

884

4

P(=O)(O[H])(OP(=O)(O[H])OP(=O)(O[H])O[H])OC([H])([H])C1([H])C([H])(C([H])(C([H])(C2C(N([H])C(N(C([H])([H])[H])C=2[H])=O)=O)O1)O[H])O[H]

OLRONOIBERDKRE-XUTVFYLZSA-N

InChI=1S/C10H17N2O15P3/c1-12-2-4(9(15)11-10(12)16)8-7(14)6(13)5(25-8)3-24-29(20,21)27-30(22,23)26-28(17,18)19/h2,5-8,13-14H,3H2,1H3,(H,20,21)(H,22,23)(H,11,15,16)(H2,17,18,19)/t5-,6-,7-,8+/m1/s1

[[(2R,3S,4R,5S)-3,4-dihydroxy-5-(1-methyl-2,4-dioxopyrimidin-5-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate

n1-methylpseudouridine-5'-triphosphate; 1428903-59-6; 1-Methylpseudouridine-5'-triphosphate; N1-.Pseudo.utp; 5ENC36P33M; N1-Methylpseudouridine-5 inverted exclamation marka-triphosphate; ((2R,3S,4R,5S)-3,4-Dihydroxy-5-(1-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)tetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate; 1-Methylpseudo-UTP;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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