Preparative Separation of Ribonucleoside Monophosphates by Ion-Pair Reverse-Phase HPLC

Structural and dynamic investigations of RNA by nuclear magnetic resonance (NMR) spectroscopy strongly benefit from isotopic-labeling strategies. Among these, nucleotide-specific and site-specific labeling methods can help tremendously in simplifying complex NMR data, while providing unique opportunities for structural investigation of larger RNAs. Such methods generally require separation of individual isotopically labeled ribonucleoside monophosphates prior to their conversion into nucleoside triphosphates and selective incorporation of these nucleoside triphosphates into the RNA. This chapter provides the experimental details for preparative separation of ribonucleoside monophosphates by ion-pair reverse-phase HPLC. It also describes a quick procedure for clean-up and quality control of the individual ribonucleoside monophosphates.