Ion Pair Reverse-Phase Chromatography: A Versatile Platform for the Analysis of RNA

Introduction

The requirement for high throughput analytical tools that can readily separate, purify and analyse ribonucleic acids (RNA) are assuming increasing significance with the recent discoveries of the diverse and important roles RNA plays in biological systems. RNA consists of a long chain of nucleotide units made up of a nitrogenous base, a ribose sugar, and a phosphate.

Cells contain three abundant classes of RNA including messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA), these can be classified as RNAs involved in protein synthesis. In addition to these abundant RNAs involved in protein synthesis a wide range of cellular RNAs exist within the cell. Our appreciation of the relative importance of RNA in numerous biological processes has increased substantially over recent years. The discoveries of catalytic RNA [1-2], RNA interference and non-coding regulatory RNAs [reviewed in 3-4], and the recent association with mediating antiviral response in prokaryotes [5-6] have all impacted a broad range of disciplines. In addition, the utilisation of RNA as a biotherapeutic tool is gaining significant importance in the field of medical research [7-8].

The emergence of RNA biotherapeutics including small interfering RNA (siRNA) therapeutics, RNA aptamers and antisense reagents which are all chemically synthesized, has led to the requirement of high throughput chromatographic procedures to purify and characterise RNA. Ion pair reverse-phase chromatography (IP RP HPLC) has been widely applied for the study of nucleic acids, in particular DNA [9-10]. More recently, a number of studies have utilised IP RP HPLC for the purification and analysis of RNA, demonstrating its versatility in a variety of different applications; from the routine purification of synthetic oligoribonucleotides, through to the analysis of complex RNA:RNA interactions. This research demonstrates that IP RP HPLC is a versatile platform for the analysis of RNA. Careful selection of the chromatography conditions including the ion pair reagent, temperature and additives to the mobile phase, facilitates the operation of the IP RP HPLC under different modes, enabling the study of a wide of range RNAs and biological systems.