N6-Methyl-dATP
目录号 : GB20075N6-Methyl-dATP作为一种碱基修饰的核糖核苷三磷酸，在豚鼠、大肠绦虫等生物体内可作为P2Y嘌呤受体的有效激动剂。
Cas No.:5085-65-4(free acid)
Sample solution is provided at 25 µL, 10mM.
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N6-Methyl-dATP, as a base-modified ribonucleoside triphosphate, can act as an effective agonist for P2Y purinergic receptors in organisms such as guinea pigs and tapeworms[1]. N6-Methyl-dATP has also been demonstrated to play a critical role in carcinogenesis and stem cell fate determination[2].
In vitro, when HeLa cells are treated with 4μM N6-Methyl-dATP for 45 minutes, N6-methyladenine (N6-mA) must first be taken up by the cells and metabolized into the active form N6-Methyl-dATP to serve as a substrate for DNA polymerase and be integrated into DNA. This process falls under the category of RNA methylation products erroneously intervening in DNA synthesis through the nucleotide salvage pathway, and its integration mechanism relies on the non-specific recognition of modified nucleotides by DNA polymerase[3].
In vivo, microinjection of 0.3 mM N6-Methyl-dATP into homozygous MTH1KO zebrafish with knockout of the mth1 gene reveals that N6-Methyl-dATP present in the nucleotide pool can be incorporated into DNA, and this process can be blocked by the MTH1 protein[4]. In Arabidopsis MAPDA mutants, a high ratio of N6-Methyl-dATP/ATP (1.49%) induces root growth defects due to erroneous integration by RNA polymerase II or interference with energy metabolism[5].
References:
[1] He ML, Koshimizu TA, Tomić M, et al. Purinergic P2X(2) receptor desensitization depends on coupling between ectodomain and C-terminal domain. Mol Pharmacol 2002, 62(5):1187-1197.
[2] Pacini CE, Bradshaw CR, Garrett NJ, et al. Characteristics and homogeneity of N6-methylation in human genomes. Sci Rep 2019, 9(1):5185.
[3] Musheev MU, Baumgärtner A, Krebs L, et al. The origin of genomic N6-methyl-deoxyadenosine in mammalian cells. Nature Chemical Biology 2020, 16(6):630-634.
[4] Scaletti ER, Vallin KS, Bräutigam L, et al. MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool. J Biol Chem 2020, 295(15):4761-4772.
[5] Chen M, Urs MJ, Sánchez-González I, et al. m(6)A RNA Degradation Products Are Catabolized by an Evolutionarily Conserved N(6)-Methyl-AMP Deaminase in Plant and Mammalian Cells. Plant Cell 2018, 30(7):1511-1522.
N6-Methyl-dATP作为一种碱基修饰的核糖核苷三磷酸，在豚鼠、大肠绦虫等生物体内可作为P2Y 嘌呤受体的有效激动剂[1]。N6-Methyl-dATP也被证明在致癌作用和干细胞命运的决定中扮演关键角色[2]。
在体外，N6-Methyl-dATP（4μM）处理HeLa细胞45 min，N6-甲基腺嘌呤（N6-mA）需先被细胞摄取并代谢为活性形式N6-Methyl-dATP，方可作为DNA聚合酶的底物整合至DNA中。这一过程属于 RNA甲基化产物通过核苷酸补救途径错误介入DNA合成的范畴，其整合机制依赖于DNA聚合酶对修饰核苷酸的非特异性识别 [3]。
在体内，N6-Methyl-dATP（0.3mM）通过显微注射至mth1基因敲除的纯合子MTH1KO斑马鱼中，发现核苷酸库中存在的N6-Methyl-dATP能够掺入DNA中，而这一过程可被MTH1蛋白阻止[4]。在拟南芥MAPDA突变体中，高比率的N6-Methyl-dATP/ATP（1.49%）诱导RNA聚合酶II错误整合或能量代谢干扰导致根系生长缺陷[5]。
| Cell experiment [1]: | |
Cell lines | HeLa cells |
Preparation Method | HeLa cells were collected, trypsin-digested, washed and lysed. The protein concentration of the cell lysate was determined by the Bradford assay. In a reaction system with components like 40mM HEPES/KOH pH 7.8, 70mM KCl, 5m MMgCl₂, 10μM ZnCl₂ and 500μM DTT, experimental groups with 20μM dATP or 16μM dATP plus 4μm N6-Methyl-dATP were set up. Template DNA, primers and HeLa cell lysate were added and incubated at 37°C for 45 minutes. After reaction, nucleic acids were purified by phenol-chloroform extraction and ethanol precipitation. Nucleotides and primers were removed by ultrafiltration and concentration. Amplification products were PAGE-purified and ethanol-precipitated. The DNA precipitate was dissolved and digested into nucleosides, and then N6-mA level was measured by LC-MS. |
Reaction Conditions | 4μM; 37°C for 45min |
Applications | In mammalian cells, DNA polymerase can utilize N6-Methyl-dATP and incorporate N6-mA into DNA. |
| Animal experiment [2]: | |
Animal models | MTH1KO zebrafish |
Preparation Method | The fertilized MTH1WT and MTH1KO zebrafish eggs were grouped. One group was microinjected with N6-methyl-dATP solution. Approximately 2nl volume of 0.3mM N6-methyl-dATP was injected into the injection buffer (9μM spermidine, 0.21μM spermidine, 0.3% phenol red) , while the other group was not injected as a control. And the eggs were distributed onto the plates of E3 medium (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2·2H2O, 0.33mM MgSO4). The viability of zebrafish embryos was evaluated 24 hours after injection, and the zebrafish were harvested 32 hours later. Zebrafish DNA from different treatment groups was isolated using the DNeasy Blood and Tissue Kit. The processed DNA samples were hydrolyzed, and various enzymes (nuclease P1, Benzonase nuclease, Escherichia coli alkaline phosphatase) were added and incubated at 37°C for 1hour. Precipitate the protein, take the supernatant and redissolve it in water. The content of N6-methyl-dATP in zebrafish embryo DNA of different treatment groups was analyzed by LC-MS/MS using the Agilent 6495 triple quadrupole LC/MS/MS system and specific chromatographic columns and mobile phase conditions. |
Dosage form | 0.3mM; 24h |
Applications | N6-methyl-dATP is incorporated into DNA in an MTH1-dependent manner. |
References: | |
| Cas No. | 5085-65-4(free acid) | SDF | Download SDF |
| 分子式 | C11H18N5O12P3 (free acid) | 分子量 | 505.2 (free acid) |
| 溶解度 | 储存条件 | Store at -20°C or below | |
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1 mg | 5 mg | 10 mg |
| 1 mM | 1.9794 mL | 9.8971 mL | 19.7941 mL |
| 5 mM | 0.3959 mL | 1.9794 mL | 3.9588 mL |
| 10 mM | 0.1979 mL | 0.9897 mL | 1.9794 mL |
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- Purity: >99.50%
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