Since the discovery of nucleotides over 100 years ago, extensive studies have revealed the importance of nucleotides for homeostasis, health and disease. However, there remains no established method to investigate quantitatively and accurately intact nucleotide incorporation into RNA and DNA. Herein, we report a new method, Stable-Isotope Measure Of Influxed Ribonucleic Acid Index (SI-MOIRAI), for the identification and quantification of the metabolic fate of ribonucleotides and their precursors. SI-MOIRAI, named after Greek goddesses of fate, combines a stable isotope-labelling flux assay with mass spectrometry to enable quantification of the newly synthesized ribonucleotides into r/m/tRNA under a metabolic stationary state. Using glioblastoma (GBM) U87MG cells and a patient-derived xenograft (PDX) GBM mouse model, SI-MOIRAI analyses showed that newly synthesized GTP was particularly and disproportionally highly utilized for rRNA and tRNA synthesis but not for mRNA synthesis in GBM in vitro and in vivo. Furthermore, newly synthesized pyrimidine nucleotides exhibited a significantly lower utilization rate for RNA synthesis than newly synthesized purine nucleotides. The results reveal the existence of discrete pathways and compartmentalization of purine and pyrimidine metabolism designated for RNA synthesis, demonstrating the capacity of SI-MOIRAI to reveal previously unknown aspects of nucleotide biology.

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SI-MOIRAI：一种识别和量化核苷酸代谢命运的新方法

自 100 多年前发现核苷酸以来，广泛的研究揭示了核苷酸对体内平衡、健康和疾病的重要性。然而，仍然没有确定的方法来定量和准确地研究完整的核苷酸掺入 RNA 和 DNA。在此，我们报告了一种新方法，即流入核糖核酸指数的稳定同位素测量 (SI-MOIRAI)，用于识别和量化核糖核苷酸及其前体的代谢命运。以希腊命运女神命名的 SI-MOIRAI 将稳定同位素标记通量测定与质谱法相结合，可以在代谢静止状态下将新合成的核糖核苷酸量化为 r/m/tRNA。使用胶质母细胞瘤 (GBM) U87MG 细胞和患者来源的异种移植物 (PDX) GBM 小鼠模型，SI-MOIRAI 分析表明，新合成的 GTP 特别且不成比例地高度用于 rRNA 和 tRNA 合成，但不用于体外和体内 GBM 中的 mRNA 合成。此外，与新合成的嘌呤核苷酸相比，新合成的嘧啶核苷酸对 RNA 合成的利用率显着降低。结果揭示了指定用于 RNA 合成的离散途径和嘌呤和嘧啶代谢的区室化的存在，证明了 SI-MOIRAI 揭示核苷酸生物学以前未知方面的能力。与新合成的嘌呤核苷酸相比，新合成的嘧啶核苷酸对 RNA 合成的利用率显着降低。结果揭示了指定用于 RNA 合成的离散途径和嘌呤和嘧啶代谢的区室化的存在，证明了 SI-MOIRAI 揭示核苷酸生物学以前未知方面的能力。与新合成的嘌呤核苷酸相比，新合成的嘧啶核苷酸对 RNA 合成的利用率显着降低。结果揭示了指定用于 RNA 合成的离散途径和嘌呤和嘧啶代谢的区室化的存在，证明了 SI-MOIRAI 揭示核苷酸生物学以前未知方面的能力。