Thermoresponsive chromatography with regulated electrostatic interactions for effective separation of nucleic acids

本研究致力于设计和表征一种新型的热响应层析系统，在该系统中，表面特性可以改变亲水/疏水性质，同时表面电荷密度可以在水环境中作为唯一变量随温度变化。这样的表面是为了有效地分离生物活性核酸而准备的，而不需要使用几个以不同分离模式工作的色谱柱。我们已经阐明了阳离子热响应型聚(N-异丙基丙烯酰胺)(PIPAAm)修饰表面的表面性质随温度的变化，包括疏水性变化和表面电位变化。用该柱基质成功地实现了腺苷核苷酸、AMP、ADP和ATP的分离。本财政年度采用阳离子热敏色谱柱，对重复次数为2-5次的腺苷寡核苷酸和胸腺嘧啶寡核苷酸进行了分离研究。热响应式…以引发剂固定硅球为引发剂，IPAAm单体与3，3-二甲氨基丙基丙烯酰胺(DMAPAA)进行自由基聚合，制备了更多的柱基质。将样品混合物进样到聚合物修饰的硅胶填充柱(4.6 mmφ，150 mm长)中，监测这些核苷酸的洗脱行为。对于非离子PIPAAM均聚物柱，无论重复单元数多少，寡核苷酸的保留时间几乎相同。相反，寡核苷酸的保留率随着阳离子、温度响应性表面上重复单元数量的增加而增加。核苷酸碱基的极性对于改变寡核苷酸在阳离子热响应柱基质上的保留非常重要。因此，胸苷寡核苷酸的保留时间比等量磷酸基团的腺苷核苷酸略长。寡核苷酸与阳离子表面的相互作用是聚电解质的多点相互作用，因此保留率随着重复单元数的增加而增加。总之，所获得的结果表明，所制备的阳离子、热响应表面可以通过识别样品分子的电荷密度和疏水性来分离寡核苷酸。较少

This research focused on the design and characterization of novel thermoresponsive chromatography system in which the surface characteristics can change hydrophilic/hydrophobic properties and, at the same time, surface charge densities by temperature as a sole variant in aqueous milieu. Such surfaces was prepared for separation of bioactive nucleic acids efficiently without use of several chromatography columns working with different separation modes. We have already elucidated the surface property alterations of the cationic thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) modified surfaces with temperature in terms of hydrophobicity changes and surface potential changes. Furthermore, separation of adenosine nucleotides, AMP, ADP, and ATP was successfully achieved with this column matrix. In this fiscal year, separation of adenosine oligonucleotides and thymidine oligonucleotides with repeating number of 2-5, were investigated using cationic, thermoresponsive column. Thermorespon … More sive column matrices were prepared by radical polymerization of IPAAm monomer and 3,3-dimethylaminopropylacrylamide (DMAPAA) on the initiator immobilized silica beads. The sample mixture was injected into polymer-modified silica packed column (4.6mmφ, 150mm length), and elution behavior of these nucleotides were monitored. For nonionic PIPAAm homopolymer column, retention times of the oligonucleotides are almost same regardless of the number of repeating units. In contrast, retention of oligonucleotides is increased with increasing number of repeating units on cationic, thermoresponsive surfaces. Polarity of the nucleotide bases is significantly important to alter oligonucleotide retention on cationic thermoresponsive column matrices. Thus the retention times of the thymidine oligonucleotides are slightly longer than those of the adenosine nucleotides with the same amount of the phosphate groups. The interaction of oligonucleotides and cationic surfaces is multi-point interaction of polyelectrolytes, and thus the retention increased with increasing the number of repeating units. In conclusion, the obtained results suggest that the prepared cationic, thermoresponsive surfaces can be used to separate oligonucleotides through the recognition of charge densities and hydrophobicity of sample molecules. Less