Deng Jianbo, Miao Xinyu, Wang Xiaotong, Wen Sheng-Ye, Zhou Zeyang, Li Shuwei, Jiang Junhong, Yang Xiaodong, Yang Shuang
Center for Clinical Mass Spectrometry, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China.
Department of General Surgery, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, China.
Anal Methods. 2025 Jan 30;17(5):964-971. doi: 10.1039/d4ay02046d.
Ribonucleic acid (RNA), essential for protein production and immune function, undergoes glycosylation, a process that attaches glycans to RNA, generating unique glycoRNAs. These glycan-coated RNA molecules regulate immune responses and may be related to immune disorders. However, studying them is challenging due to RNA's fragility. Therefore, a robust method for identifying glycoRNA is important. To address this, we optimized parameters for RNA stability, oxidation, and digestion, thereby enriching and identifying glycoRNAs. This breakthrough paves the way for exploring their potential interactions with immune receptors and tumor suppression. Our approach involved investigating factors such as preservation reagent, enzyme buffer, digestion temperature, oxidant, glycosidase, and incubation time. We successfully optimized digestion conditions, achieving efficient cleavage of -linked glycoRNAs at room temperature using 25 mM ammonium bicarbonate, demonstrating the effectiveness of this method. Additionally, RNA preservation in RNAlater at -80 °C allows controlled release of glycoRNAs within hours. While sequential digestion of different glycoRNA types is possible, significant degradation occurs after the first glycosidase step. Therefore, we recommend separate harvesting for each glycoRNA type. We also established RNA-seq analysis for identifying various glycoRNA types, including snoRNAs and tRNAs. The optimized SPCgRNA method paves the way for further research on -glycosylation in health and disease.
核糖核酸（RNA）对蛋白质合成和免疫功能至关重要，它会经历糖基化过程，即把聚糖附着到RNA上，从而产生独特的糖基化RNA（glycoRNA）。这些糖基化修饰的RNA分子调节免疫反应，可能与免疫紊乱有关。然而，由于RNA的脆弱性，对其进行研究具有挑战性。因此，一种可靠的鉴定糖基化RNA的方法很重要。为了解决这个问题，我们优化了RNA稳定性、氧化和消化的参数，从而富集和鉴定糖基化RNA。这一突破为探索它们与免疫受体的潜在相互作用以及肿瘤抑制作用铺平了道路。我们的方法涉及研究诸如保存试剂、酶缓冲液、消化温度、氧化剂、糖苷酶和孵育时间等因素。我们成功优化了消化条件，使用25 mM碳酸氢铵在室温下实现了对连接型糖基化RNA的有效切割，证明了该方法的有效性。此外，将RNA保存在-80°C的RNAlater中可在数小时内实现糖基化RNA的可控释放。虽然可以对不同类型的糖基化RNA进行顺序消化，但在第一个糖苷酶步骤后会发生显著降解。因此，我们建议对每种糖基化RNA类型进行单独收集。我们还建立了用于鉴定各种糖基化RNA类型（包括小核仁RNA和转运RNA）的RNA测序分析方法。优化后的SPCgRNA方法为进一步研究健康和疾病中的糖基化铺平了道路。
