Diastereomer Characterization of PS-Modified Synthetic Oligonucleotides Using Cyclic IMS-MS,Analytical Chemistry

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Diastereomer Characterization of PS-Modified Synthetic Oligonucleotides Using Cyclic IMS-MS
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-09-09 , DOI: 10.1021/acs.analchem.4c02836
Edie M Sharon ₁ , Sarah M O'Keefe ₁ , Kathleen T Grassmyer ₂ , Shannon A Raab ₃ , Todd D Maloney ₂ , David E Clemmer ₁

Affiliation

Synthetic oligonucleotides have emerged as effective therapeutics that regulate gene expression to treat and prevent diseases. Oligonucleotide therapeutics are often modified with a substitution of a phosphorothioate (PS) linkage along the phosphodiester backbone to improve the drug performance and stability. The PS modification creates a mixture of diastereomer structures, increasing by a factor of 2ⁿ where n is the number of PS linkages. Despite recent draft guidances highlighting the importance of their characterization, analytical methods to measure the resulting diastereomers are currently lacking. Here, we present a method combining tandem mass spectrometry (MS) and tandem ion mobility spectrometry (IMS) using a cyclic IMS-MS instrument to study diastereomers in PS-modified oligonucleotides. This approach requires no enzymatic digestion as the intact oligonucleotides are directly injected into the MS instrument. Analogous to top-down proteomics, MS fragmentation of the intact oligonucleotide results in 3′ and 5′ fragment ends that have fewer diastereomers than their intact counterpart. Tandem IMS allows for mobility resolution of the diastereomers at the terminal ends. We tested four model oligonucleotides that differ in either the number of PS bonds or sequence to demonstrate the capability of this method to elucidate diastereomer structures on modified oligonucleotides.

中文翻译：

使用循环 IMS-MS 表征 PS 修饰的合成寡核苷酸的非对映异构体

合成寡核苷酸已成为调节基因表达以治疗和预防疾病的有效疗法。寡核苷酸治疗剂通常通过沿磷酸二酯主链替换硫代磷酸酯 (PS) 连接进行修饰，以提高药物性能和稳定性。 PS 修饰产生了非对映异构体结构的混合物，增加了 2 ⁿ倍，其中n是 PS 连接的数量。尽管最近的指南草案强调了其表征的重要性，但目前仍缺乏测量所得非对映异构体的分析方法。在这里，我们提出了一种结合串联质谱 (MS) 和串联离子迁移谱 (IMS) 的方法，使用循环 IMS-MS 仪器来研究 PS 修饰寡核苷酸中的非对映异构体。这种方法不需要酶消化，因为完整的寡核苷酸直接注射到 MS 仪器中。与自上而下的蛋白质组学类似，完整寡核苷酸的 MS 片段化会导致 3' 和 5' 片段末端的非对映体数量少于其完整对应物。 Tandem IMS 允许在终端对非对映异构体进行迁移率解析。我们测试了 PS 键数量或序列不同的四种模型寡核苷酸，以证明该方法能够阐明修饰寡核苷酸上的非对映异构体结构。

更新日期：2024-09-09

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