Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase
Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase
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DOI：
10.1007/s00216-020-02749-8
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发表时间：
2020-06-11
影响因子：
4.3
通讯作者：
Kanazawa, Hideko
中科院分区：
文献类型：
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作者：
Maekawa, Yutaro;Yamazaki, Kaichi;Kanazawa, Hideko
寡核苷酸疗法对医疗保健做出了显着贡献，非常适合治疗难以使用常规药物方式治疗的难治性疾病。然而，由于寡核苷酸分析的常用技术依赖于反相或离子交换液相色谱，因此使用有毒有机溶剂和/或离子配对试剂，因此高度寻求更好的替代品。聚（N-异丙基丙烯酰胺）（PNIPAAm）被广泛用于温度响应色谱（TRC）中，其依赖于柱温变化来控制固定相的物理性质，并且不同于常规的反相液相色谱，避免了使用有毒有机溶剂和复杂的梯度方法。在本文中，PNIPAAm共聚物水凝胶改性的二氧化硅珠用于通过TRC同时分析多个合成寡核苷酸，以识别单个核苷酸的长度、单个碱基和硫代磷酸酯化位点的数量的差异。在所有情况下均观察到温度响应性洗脱。多种寡核苷酸的所有组合的每次分离在高于较低临界溶液温度的较高温度下更好，并且在不使用有机溶剂和梯度方法的情况下进行。在多硫代磷酸化的寡核苷酸的情况下，良好的分离，实现了使用水性溶剂和等度洗脱在离子配对试剂的情况下。因此，开发的程序被认为非常适合寡核苷酸分析。图形摘要
Oligonucleotide therapeutics have contributed remarkably to healthcare, being well suited for the treatment of intractable diseases that are difficult to approach using conventional drug modalities. However, as common techniques of oligonucleotide analysis rely on reversed-phase or ion-exchange liquid chromatography and thus employ toxic organic solvents and/or ion-pairing reagents, better alternatives are highly sought after. Poly(N-isopropylacrylamide) (PNIPAAm) is widely used in temperature-responsive chromatography (TRC), which relies on column temperature variation to control the physical properties of the stationary phase and, unlike conventional reversed-phase liquid chromatography, avoids the use of toxic organic solvents and complicated gradient methods. Herein, PNIPAAm copolymer hydrogel-modified silica beads were used for the simultaneous analysis of multiple synthetic oligonucleotides by TRC to recognize differences in the length of single nucleotides, single bases, and the number of phosphorothioated sites. Temperature-responsive elution was observed in all cases. Each separation of all combinations of multiple oligonucleotides was better at higher temperatures above the lower critical solution temperature and was performed without the use of organic solvents and gradient methods. In the case of multiply phosphorothioated oligonucleotides, good separation was achieved using an aqueous solvent and isocratic elution in the absence of ion-pairing reagents. Thus, the developed procedure was concluded to be well suited for oligonucleotide analysis.Graphical abstract