Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase

Abstract

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 temperatureresponsive 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.

Effect of pore diameter on the elution behavior of analytes from thermoresponsive polymer grafted beads packed columns

“…The chromatography system uses all-aqueous mobile phases, although the addition of a small amount of methanol is required to improve detection with mass spectroscopy 28 . To improve the performance of chromatography systems, various types of PNIPAAm copolymer have been investigated such as the copolymerization of hydrophobic monomers to enhance hydrophobic interaction 29 – 32 and the copolymerization of ionic monomers to utilize electrostatic interaction besides hydrophobic interaction 33 – 37 . Moreover, several types of PNIPAAm configuration on silica bead surfaces have been investigated using various polymerization methods 4 , 38 – 40 .…”

A Molecular Shape Recognitive HPLC Stationary Phase Based on a Highly Ordered Amphiphilic Glutamide Molecular Gel

“…We also demonstrated the separation of eight steroid compounds having different chemical structures and different numbers of hydroxyl groups. Isolation and purification of biological molecules such as peptides, proteins, saccharides and nucleosides are indispensable technology in the fields of proteomics, genomics, and glycomics [40][41][42]. Further study for the separation of bio-related molecules using Sil−VPG 15 is currently underway and will be reported in the future.…”

Effective Separation for New Therapeutic Modalities Utilizing Temperature-responsive Chromatography

“…In conventional RPLC, the separation of S-oligos require an organic solvent with an ion-pairing reagent, such as tetraethylamine-acetic acid, as the mobile phase and gradient elution. In contrast, Maekawa et al reported recently that good separation of multiple phosphorothioated oligonucleotides was obtained by TR-HPLC using a P(NIPAAm-co-BMA-co-DMAPAAm) hydrogel-modified column with an aqueous solvent and an isocratic elution without ion-pairing reagents 62 .…”

“…The retention of oligonucleotides was increased by increasing the temperature, opposite to the behavior exhibited by conventional RPLCs. The separation of single nucleobases in oligonucleotides, such as guanine (G), cytosine (C), thymine (T), or adenine (A), was also achieved using the P(NIPAAm-co-BMA-co-DMAPAAm) hydrogel-modified column 62 .…”