Single-nucleotide resolution of RNAs up to 59 nucleotides by high-performance liquid chromatography

Ion-pair, reverse-phase high-performance liquid chromatography (HPLC) is a standard analytical platform for separating, purifying, and analyzing RNAs. However, a single-nucleotide resolution by using HPLC is currently limited to RNAs shorter than 25 nucleotides (nt). Here we describe a method of separating three RNA aptamers with 57, 58, and 59 nt on an XBridge ion-pair, reverse-phase HPLC column by a single-nucleotide resolution. Under a similar condition, we also show the capability of our method to resolve two structurally different, yet sequence or mass identical, 59-nt aptamers. We establish that the optimal condition to achieve a single-nucleotide resolution correlates to 50 °C and zero magnesium concentration in mobile phases. The ion-pairing agent, the buffer, and the solvent we use are also compatible for post-HPLC analysis such as mass spectrometry. Therefore, our method provides a new way of detecting, analyzing, and separating RNAs by conformation or structure and extends the ability to separate RNAs that are longer than 25 nt by single-nucleotide resolution.