With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography–mass spectrometry (LC–MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine–two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5′ cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC–MS/MS.

中文翻译：

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人 RNase 4 通过 LC–MS/MS 改善 mRNA 序列表征

随着基于合成信使 RNA (mRNA) 的疗法和疫苗的快速发展，开发用于表征长而复杂的 RNA 的分析工具变得至关重要。串联液相色谱-质谱 (LC-MS/MS) 允许直接评估 mRNA 一级序列及其修饰，而无需转化为 cDNA 或扩增。它依赖于用位点特异性内切核糖核酸酶消化 mRNA 来生成短寡核苷酸池，然后可以进行基于 MS 的序列分析。在这里，我们表明，与基准酶 RNase T1 相比，尿苷特异性人核糖核酸内切酶 hRNase 4 通过产生更多的可独特定位的裂解产物，提高了 mRNA 序列覆盖率。我们部署了 hRNase 4 来表征完全被 1-甲基假尿苷 (m1Ψ) 或 5-甲氧基尿苷 (mo5U) 取代的 mRNA，以及选择性去除尿苷的 mRNA——这是降低合成 mRNA 免疫原性的两种关键策略。最后，我们证明 hRNase 4 能够直接评估 5' 帽掺入体外转录的 mRNA。总的来说，这项研究强调了 hRNase 4 通过 LC-MS/MS 检测 mRNA 序列、身份和修饰的能力。