High resolution chromatography of ribonucleosides and its application to RNA analysis

A simple and precise method was developed for the separation of nucleosides including modified nucleosides and oligonucleotides. Nineteen kinds of nucleosides were completely separated by HPLC using an ODS column (TSK-gel ODS 80TM) and aqueous mobile phases. The RNA molecule was digested by base restrictive RNase (RNase A, RNase T1) and the digests were separated chromatographically into each oligonucleotide. The nucleoside composition of an oligonucleotide was then determined by this analytical system. It is thus possible to fit the oligonucleotide in the original RNA molecule by using modified bases as markers. The reaction site of quinacrine mustard for tRNA(Phe) (from yeast) could be determined by this analytical system.