Therapeutic messenger RNAs (mRNAs) have emerged as powerful tools in the treatment of complex diseases, especially for conditions that lack efficacious treatment. The successful application of this modality can be attributed to its ability to encode entire proteins. While the large nature of these molecules has supported their success as therapeutics, its extended size creates several analytical challenges. To further support therapeutic mRNA development and its deployment in clinical trials, appropriate methods to support their characterization must be developed. In this review, we describe current analytical methods that have been used in the characterization of RNA quality, identity, and integrity. Advantages and limitations from several analytical techniques ranging from gel electrophoresis to liquid chromatography–mass spectrometry and from shotgun sequencing to intact mass measurements are discussed. We comprehensively describe the application of analytical methods in the measurements of capping efficiency, poly A tail analysis, as well as their applicability in stability studies.

中文翻译：

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mRNA 疗法的表征

治疗性信使 RNA (mRNA) 已成为治疗复杂疾病的强大工具，特别是对于缺乏有效治疗的疾病。这种模式的成功应用可归因于其编码整个蛋白质的能力。虽然这些分子的巨大性质支持了它们作为治疗药物的成功，但其尺寸的扩大带来了一些分析挑战。为了进一步支持治疗性 mRNA 的开发及其在临床试验中的部署，必须开发适当的方法来支持其表征。在这篇综述中，我们描述了当前用于表征 RNA 质量、同一性和完整性的分析方法。讨论了从凝胶电泳到液相色谱-质谱分析以及从鸟枪测序到完整质量测量等几种分析技术的优点和局限性。我们全面描述了分析方法在加帽效率测量、多聚腺苷酸尾部分析中的应用，以及它们在稳定性研究中的适用性。