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

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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