Separations R&D, Waters Corporation, Milford, Massachusetts 01757, United States.
Discovery and Development, Waters Corporation, Milford, Massachusetts 01757, United States.
Anal Chem. 2023 Sep 26;95(38):14308-14316. doi: 10.1021/acs.analchem.3c02552. Epub 2023 Sep 11.
Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.
信使 RNA（mRNA）是一类新型的治疗性化合物。目前，mRNA 技术的进步需要开发有效的分析方法。在这项工作中，我们描述了几种测量 mRNA 多聚（A）尾长度和异质性的方法的开发。首先，用 RNase T1 酶从 mRNA 中切割多聚（A）尾。用排阻层析法分析释放的多聚（A）尾的平均长度。用高分辨率离子对反相液相色谱（IP RP LC）法估计多聚（A）尾的大小异质性。IP RP LC 法可分辨长达 150 个核苷酸的多聚（A）尾寡核苷酸变体。这两种方法都使用一种稳健的紫外检测方法，适用于质量控制实验室中的 mRNA 分析。通过对同一 mRNA 样本的 LC-MS（LC-MS）分析，对结果进行了验证。多聚（A）尾长度和异质性的结果吻合较好。
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