HPLC Retention Behavior of Oligonucleotides on an Anion-Exchange HPLC with a High Concentration of Urea

Roles of short RNA molecules in the emergence process of primitive life-like system and physiological activities have been extensively studied from the viewpoint of both fundamental and applicational areas. The discovery of catalytic functions of short RNA molecules suggests that the most primitive life-like systems have emerged from a chemical network consisting of short functional RNA molecules that existed. However, the quantitative analysis of such short RNA, including ribozymes, micro-RNA, and small non-coding RNA consisting of less than approximately 100 nucleotide units is difficult to perform using conventional techniques, such as gel electrophoresis (GE) and high-performance liquid chromatography (HPLC). Both GE and HPLC are complementarily used for different purposes related to oligonucleotide analysis. Recently, the HPLC method’s usefulness with a high urea concentration has been demonstrated for short oligonucleotides. In this study, the HPLC separation behavior in the presence of a high urea concentration is studied using a Tosoh DNA-NPR anion-exchange column (diameter 2.0 mm, length 100 mm along with a guard column) with a buffer containing 0.02 M Tris and 7.5 M urea at pH 9.0 and a buffer containing 1.5 M NaCl, 0.02 M Tris, and 7.5 M urea at pH 9.0 mixed with a linear gradient at a flow rate of 0.15 mL min⁻¹ at 35 °C. Different sequences of oligonucleotides are collected to know how the influence of secondary structure formation can be reduced. Molecular modeling of the secondary structure is applied to evaluate the HPLC retention behaviors of oligonucleotides on an anion-exchange column. This study provides valuable information for selecting the elution conditions for separating the RNA and DNA oligonucleotides and preheating treatment.