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| Targets |
5-Azidomethyl-uridine does not target a specific biological receptor or enzyme; rather, it is a metabolic label that is incorporated into newly synthesized RNA. The compound is cell-permeable and is converted to the triphosphate form (5-Azidomethyl-UTP) by cellular uridine kinase and nucleotide kinases. The triphosphate is then utilized by RNA polymerases as a substrate, resulting in the incorporation of 5-azidomethyl-uridine into nascent RNA transcripts. Once incorporated, the azide group serves as a bioorthogonal handle for subsequent conjugation with alkyne-functionalized probes via copper-catalyzed azide-alkyne cycloaddition (CuAAC, “click chemistry“). This allows visualization, capture, or sequencing of newly synthesized RNA. The mechanism does not involve inhibition of a biological pathway but rather the hijacking of the natural RNA synthesis machinery for labeling purposes.
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| ln Vitro |
5-Azidomethyl-uridine is not an active pharmaceutical ingredient with intrinsic biological activity; it is a chemical tool for studying RNA dynamics. In vitro, the compound is used in cell-free transcription assays to produce labeled RNA. In a typical assay, T7 RNA polymerase is incubated with a DNA template, NTPs (ATP, CTP, GTP, and UTP), and 5-Azidomethyl-uridine (substituting a fraction of UTP). The transcribed RNA incorporates the modified uridine. The RNA can then be detected or captured via click chemistry with alkyne-biotin or alkyne-fluorophores. The compound does not inhibit RNA polymerase activity at concentrations up to 100 uM. In cellular systems, 5-Azidomethyl-uridine is non-toxic at labeling concentrations (typically 0.1-1 mM). The labeled RNA can be visualized by fluorescence microscopy after click reaction, enabling the study of RNA synthesis dynamics, localization, and turnover. The compound is also used for nascent RNA sequencing (e.g., “TimeLapse-seq“).
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| ln Vivo |
A non-cellular protocol for using 5-Azidomethyl-uridine involves its incorporation into RNA in a cell-free transcription system. A typical 20 uL reaction contains 40 mM Tris-HCl (pH 8.0), 25 mM MgCl2, 10 mM DTT, 2 mM spermidine, 1 unit/uL RNasin, 2 mM each of ATP, CTP, GTP, and 1-2 mM UTP, and 0.5-1 mM 5-Azidomethyl-uridine. The DNA template (1 ug of linearized plasmid or PCR product containing a T7 promoter) and 20 units of T7 RNA polymerase are added. The mixture is incubated at 37degC for 2-4 hours. The RNA is then purified using phenol-chloroform extraction and ethanol precipitation. The 5-azidomethyl-uridine-labeled RNA is detected by click chemistry: the RNA (1 ug) is incubated with 100 uM biotin-alkyne or a fluorescent alkyne dye (e.g., Cy5-alkyne), 1 mM CuSO4, 2 mM TCEP, and 100 uM THPTA ligand in PBS for 1 hour at 37degC. The labeled RNA is purified and analyzed by gel electrophoresis, Northern blot, or mass spectrometry.
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| Enzyme Assay |
A typical in vitro cellular protocol for using 5-Azidomethyl-uridine to label nascent RNA involves the incorporation of the compound into newly synthesized RNA in living cells. HeLa or other adherent cells are seeded in 6-well plates (1×10⁵ cells/well) in DMEM with 10% FBS and cultured at 37degC in 5% CO2 for 24 hours. The medium is replaced with fresh medium containing 0.5-1 mM 5-Azidomethyl-uridine. Cells are incubated for 30-120 minutes to allow incorporation of the modified uridine into RNA. For pulse-chase experiments, cells are washed with PBS and incubated in normal medium for additional time points (e.g., 1, 2, 4, 8 hours). For visualization, cells are washed with PBS, fixed with 4% paraformaldehyde in PBS for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes. Click chemistry is performed using 5 uM alkyne-fluorophore (e.g., Alexa Fluor 488-alkyne), 1 mM CuSO4, 2 mM TCEP, and 100 uM THPTA in PBS for 30 minutes at room temperature. Nuclei are counterstained with DAPI. Cells are imaged by fluorescence microscopy. To measure total RNA labeling, cells are harvested, and RNA is isolated using TRIzol. The purified RNA (1-5 ug) is subjected to click chemistry with biotin-alkyne, followed by dot blot analysis using streptavidin-HRP.
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| Cell Assay |
An in vivo animal protocol for using 5-Azidomethyl-uridine is not common for research due to potential toxicity and stability concerns, but it may be used for labeling RNA in whole organisms. For example, in a mouse model, 5-Azidomethyl-uridine (50-200 mg/kg) can be administered via intraperitoneal (IP) injection. After a labeling period of 1-4 hours, the animal is euthanized, and tissues (e.g., liver, brain, spleen) are harvested. RNA is extracted from tissues using TRIzol. Purified RNA is then subjected to click chemistry with biotin-alkyne or fluorophore-alkyne for downstream detection or sequencing. For imaging, mice may be perfused with fixative, and tissue sections (e.g., brain slices) are processed for fluorescence microscopy after click labeling. This approach allows spatial mapping of active transcription. However, due to the potential for incorporation into DNA (upon conversion to deoxynucleotides), careful controls are required. The compound is not intended for human use and is for research purposes only.
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| Animal Protocol |
The pharmacokinetic (PK) properties of 5-Azidomethyl-uridine are not well characterized, as it is a research reagent rather than a drug candidate. The compound is cell-permeable and is expected to be taken up by cells via nucleoside transporters (e.g., ENT1, ENT2). Once inside the cell, it is phosphorylated by uridine-cytidine kinase (UCK2) to the monophosphate, then to the diphosphate and triphosphate forms. The triphosphate is incorporated into RNA by RNA polymerases. The compound is likely metabolized and cleared rapidly, but formal PK studies are not available. The compound has a molecular weight of 299.24 and is soluble in DMSO (125 mg/mL) and water (approximately 5 mg/mL). For in vivo studies, the compound is typically formulated in PBS or saline. The half-life in plasma and tissues is expected to be short (minutes to hours). Detailed ADME data are not published.
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| ADME/Pharmacokinetics |
Toxicity data for 5-Azidomethyl-uridine are limited. The compound is a modified nucleoside and is not considered highly toxic at concentrations typically used for RNA labeling (0.1-1 mM in cell culture, 50-200 mg/kg in animals). In cell culture, viability remains >90% after 2-4 hours of treatment with up to 1 mM of the compound. In animal studies, doses up to 200 mg/kg have been used without reports of acute toxicity, but formal LD50 and chronic toxicity studies have not been published. Standard laboratory safety precautions should be followed when handling 5-Azidomethyl-uridine, including the use of gloves, lab coats, and safety glasses. The compound contains an azide group, which can be potentially explosive in pure form or at high concentrations; it should be handled with care, kept away from reducing agents, and stored as a solution or solid in a cool, dry place. Avoid heating, shock, or friction. It is for research use only and is not intended for human therapeutic or diagnostic applications.
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| Toxicity/Toxicokinetics |
5-Azidomethyl-uridine is a click chemistry reagent used for RNA labeling and nascent RNA synthesis studies. It contains an azide group that undergoes copper-catalyzed azide-alkyne cycloaddition (CuAAC) with alkyne-conjugated probes. The compound is cell-permeable and is incorporated into newly transcribed RNA, allowing visualization, capture, and sequencing of nascent transcripts. It is used as an alternative to 5-ethynyluridine (5-EU) and 5-bromouridine (BrU) for measuring RNA synthesis in proliferating cells. The molecular formula is C10H13N5O6, and the molecular weight is 299.24. The compound is soluble in DMSO (125 mg/mL) and water. 5-Azidomethyl-uridine is valuable in synthetic biology, nucleic acid therapeutics, and molecular probe development for exploring RNA structure, dynamics, and interactions. It is for research use only and is not approved for clinical applications.
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| References |
| Molecular Formula |
C10H13N5O6
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| Molecular Weight |
299.24
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| Exact Mass |
299.086
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| CAS # |
24751-67-5
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| PubChem CID |
121487445
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| Appearance |
White to off-white solid powder
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| LogP |
-1.07
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
21
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| Complexity |
530
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| Defined Atom Stereocenter Count |
4
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| SMILES |
C1=C(C(=O)NC(=O)N1[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O)CN=[N+]=[N-]
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| InChi Key |
ZAFBFGDCEFQDFD-JXOAFFINSA-N
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| InChi Code |
InChI=1S/C10H13N5O6/c11-14-12-1-4-2-15(10(20)13-8(4)19)9-7(18)6(17)5(3-16)21-9/h2,5-7,9,16-18H,1,3H2,(H,13,19,20)/t5-,6-,7-,9-/m1/s1
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| Chemical Name |
5-(azidomethyl)-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dione
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO: 125 mg/mL (417.72 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.95 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.95 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (6.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3418 mL | 16.7090 mL | 33.4180 mL | |
| 5 mM | 0.6684 mL | 3.3418 mL | 6.6836 mL | |
| 10 mM | 0.3342 mL | 1.6709 mL | 3.3418 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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