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

寡核苷酸疗法对医疗保健做出了显着贡献，非常适合治疗难于使用常规药物治疗方法治疗的顽固性疾病。但是，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，因此采用有毒的有机溶剂和/或离子对试剂，因此，人们一直在寻求更好的替代方法。聚（N-异丙基丙烯酰胺（PNIPAAm）被广泛用于温度响应色谱（TRC）中，该色谱依靠柱温变化来控制固定相的物理性质，并且与传统的反相液相色谱法不同，它避免了使用有毒的有机溶剂和复杂的渐变方法。在本文中，PNIPAAm共聚物水凝胶修饰的硅胶珠用于通过TRC同时分析多个合成寡核苷酸，以识别单个核苷酸的长度，单个碱基的长度和硫代磷酸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。在高于较低的临界溶液温度的较高温度下，多个寡核苷酸的所有组合的每次分离效果更好，并且无需使用有机溶剂和梯度方法即可进行分离。在使用多种硫代磷酸化寡核苷酸的情况下，使用水性溶剂和在不存在离子对试剂的情况下进行等度洗脱可实现良好的分离。因此，结论是所开发的方法非常适合寡核苷酸分析。

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