 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C10H13LI4N2O15P3 |
| Molecular Weight | 521.90 |
| Related CAS # | N1-Methylpseudouridine-5′-triphosphate trisodium;N1-Methylpseudouridine-5′-triphosphate;1428903-59-6 |
| Appearance | Typically exists as solid at room temperature |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Nucleobase-modified nucleotide for synthesis of mRNA |
| ln Vitro | 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]. |
| Enzyme Assay |
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. 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. 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. |
| Cell Assay |
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. 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. |
| Animal Protocol |
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. 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. |
| References | [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. |
Solubility Data
| Solubility (In Vitro) | 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 |
| Solubility (In Vivo) |
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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9161 mL | 9.5804 mL | 19.1608 mL | |
| 5 mM | 0.3832 mL | 1.9161 mL | 3.8322 mL | |
| 10 mM | 0.1916 mL | 0.9580 mL | 1.9161 mL |