Expanding the Analytical Toolbox for the Nondenaturing Analysis of siRNAs with Salt-Mediated Ion-Pair Reversed-Phase Liquid Chromatography

IF 6.7 1区化学 Q1 CHEMISTRY, ANALYTICAL

Analytical Chemistry Pub Date : 2024-11-11 DOI:10.1021/acs.analchem.4c05248

Martin Enmark, Ilaria Furlan, Porya Habibollahi, Christian Manz, Torgny Fornstedt, Jörgen Samuelsson, Eivor Örnskov, Manasses Jora

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引用次数: 0

Abstract

Short interfering RNA (siRNA) represents a rapidly expanding class of marketed oligonucleotide therapeutics. Due to its double-stranded nature, the characterization of siRNA is twofold: (i) at the single-strand (denaturing) level for impurity profiling and (ii) at the intact (nondenaturing) level to confirm duplex formation and quantify excess single strands (including single strand-derived impurities). While denaturing analysis can be carried out using conventional ion-pair reversed-phase liquid chromatography (IP-RPLC), nondenaturing characterization of siRNA is a significantly less straightforward task. Typical IP-RPLC conditions have an intrinsic denaturing effect on siRNA, thereby limiting the development of viable approaches for the intact duplex analysis. In this study, we demonstrate, through the design of experiments of siRNA melting temperatures and chromatography analyses, that the simple addition of salts, such as phosphate-buffered saline and ammonium acetate, to eluents enhances the suitability of IP-RPLC for the nondenaturing analysis of siRNA during both UV- and mass spectrometry-based analysis. This work represents a milestone in overcoming the challenges associated with nondenaturing analysis of siRNAs by IP-RPLC and offers a fresh angle for exploring IP-RPLC of siRNAs.

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利用盐介导离子对反相液相色谱法扩展 siRNA 非变性分析工具箱

短干扰 RNA（siRNA）是市场上迅速发展的一类寡核苷酸疗法。由于其双链性质，siRNA 的表征包括两个方面：(i) 单链（变性）水平，用于杂质分析；(ii) 完整（非变性）水平，用于确认双链的形成和量化多余的单链（包括单链衍生的杂质）。变性分析可使用传统的离子对反相液相色谱法（IP-RPLC）进行，而 siRNA 的非变性表征则不那么简单。典型的 IP-RPLC 条件对 siRNA 有固有的变性作用，因此限制了完整双链分析可行方法的开发。在本研究中，我们通过设计 siRNA 熔化温度和色谱分析实验证明，在洗脱液中简单添加磷酸盐缓冲盐水和醋酸铵等盐类，可提高 IP-RPLC 在基于紫外和质谱分析的 siRNA 非变性分析中的适用性。这项工作是克服用 IP-RPLC 进行 siRNA 非变性分析所面临的挑战的一个里程碑，为探索 siRNA 的 IP-RPLC 提供了一个新的角度。

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来源期刊

Analytical Chemistry 化学-分析化学

CiteScore

12.10

自引率

12.20%

发文量

1949

审稿时长

1.4 months

期刊介绍： Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.