title: Single-nucleotide resolution of RNAs up to 59 nucleotides by high-performance liquid chromatography.
abstract: 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 59nt 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 25nt by single-nucleotide resolution.
authors.affiliation: Department of Chemistry, Center for Neuroscience Research, University at Albany, State University of New York (SUNY), Albany, NY 12222, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Huang
authors.forename: Sabarinath
authors.initials: S
authors.lastname: Jayaseelan
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hebert
authors.forename: Hyojung
authors.initials: H
authors.lastname: Seo
authors.forename: Li
authors.initials: L
authors.lastname: Niu
mesh_terms: D052157:Aptamers, Nucleotide / Q000032:analysis / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D008274:Magnesium / Q000737:chemistry; D009711:Nucleotides / Q000032:analysis / Q000302:isolation & purification*; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D052157:Aptamers, Nucleotide; D009711:Nucleotides; D012313:RNA; D008274:Magnesium