Established bottom-up approaches for the characterization of nucleic acids (NAs) rely on the strand-cleavage activity of nucleotide-specific endonucleases to generate smaller oligonucleotides amenable to gas-phase sequencing. The complexity of these hydrolytic mixtures calls for the utilization of a front-end separation to facilitate full mass spectrometric (MS) characterization. This report explored the merits of microfluidic capillary zone electrophoresis (CZE) as a possible alternative to common liquid chromatography techniques. An oligonucleotide ladder was initially employed to investigate the roles of fundamental analyte features and experimental parameters in determining the outcome of CZE-MS analyses. The results demonstrated the ability to fully resolve the various rungs into discrete electrophoretic peaks with full-width half-height (FWHH) resolution that was visibly affected by the overall amount of material injected into the system. Analogous results were obtained from a digestion mixture prepared by treating yeast tRNA^Phe (75 nt) with RNase T1, which provided several well-resolved peaks in spite of the increasing sample heterogeneity. The regular shapes of such peaks, however, belied the fact that most of them contained sets of comigrating species, as shown by the corresponding MS spectra. Even though it was not possible to segregate each species into an individual electrophoretic peak, the analysis still proved capable of unambiguously identifying a total of 29 hydrolytic products, which were sufficient to cover 96% of the tRNA^Phe’s sequence. Their masses accurately reflected the presence of modified nucleotides characteristic of this type of substrate. The analysis of a digestion mixture obtained from the 364 nt HIV-1 5′-UTR proved to be more challenging. The electropherogram displayed fewer well-resolved peaks and significantly greater incidence of product comigration. In this case, fractionating the highly heterogeneous mixture into discrete bands helped reduce signal suppression and detection bias. As a result, the corresponding MS data enabled the assignment of 248 products out of the possible 513 predicted from the 5′-UTR sequence, which afforded 100% sequence coverage. This figure represented a significant improvement over the 36 total products identified earlier under suboptimal conditions, which afforded only 57% coverage, or the 83 observed by direct infusion nanospray-MS (72%). These results provided a measure of the excellent potential of the technique to support the bottom-up characterization of progressively larger NA samples, such as putative NA therapeutics and mRNA vaccines.

中文翻译：

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研究微流控毛细管区带电泳-质谱 (CZE-MS) 在较大 RNA 自下而上表征中的优点

用于表征核酸 (NA) 的自下而上方法依赖于核苷酸特异性核酸内切酶的链切割活性来生成适合气相测序的较小寡核苷酸。这些水解混合物的复杂性要求利用前端分离来促进完整的质谱 (MS) 表征。本报告探讨了微流控毛细管区带电泳 (CZE) 作为普通液相色谱技术的可能替代方案的优点。寡核苷酸梯最初用于研究基本分析物特征和实验参数在确定 CZE-MS 分析结果中的作用。结果表明，能够将各个梯级完全解析为具有全宽半高 (FWHH) 分辨率的离散电泳峰，该分辨率明显受到注入系统的材料总量的影响。^{通过用 RNase T1 处理酵母 tRNA Phe} (75 nt)制备的消化混合物获得了类似的结果，尽管样品异质性不断增加，但仍提供了几个分辨率良好的峰。然而，这些峰的规则形状掩盖了这样一个事实，即它们中的大多数包含共迁移物质组，如相应的 MS 谱所示。尽管不可能将每个物种分离成单独的电泳峰，但该分析仍然证明能够明确识别总共 29 种水解产物，这些产物足以覆盖 tRNA ^Phe序列的 96%。它们的质量准确地反映了此类底物特有的修饰核苷酸的存在。事实证明，对从 364 nt HIV-1 5'-UTR 获得的消化混合物进行分析更具挑战性。电泳图显示出较少的清晰峰，并且产物共迁移的发生率明显较高。在这种情况下，将高度异质的混合物分成离散的频带有助于减少信号抑制和检测偏差。结果，相应的 MS 数据能够从 5'-UTR 序列预测的 513 个可能产物中分配出 248 个产物，从而提供 100% 的序列覆盖率。这一数字表明，较之前在次优条件下确定的 36 种产品（覆盖率仅为 57%）或直接输注纳喷雾 MS 观察到的 83 种产品（72%）相比，有了显着改善。这些结果衡量了该技术在支持逐渐增大的 NA 样本（例如假定的 NA 疗法和 mRNA 疫苗）的自下而上表征方面的巨大潜力。