Methods for Sequencing Oligoribonucleotides and RNA by High Performance Liquid Chromatography1

Conditions were established for the separation and quantitative determination of ribonucleo-sides, mono- and oligo-ribonucleotides by high-performance liquid chromatography (HPLC) on columns of AS-Pellionex SAX and AL-Pellionex WAX. By combining a high-speed UV spectrum monitor with an HPLC apparatus, products of RNase digestions of oligo-nucleotides and 5S ribosomal RNA (rRNA) were identified by measuring their UV spectra under continuous solvent flow, and also from their retention times on the columns (positions of elution). It took only 10 to 30 min for one chromatography run and required less than 0.01 Auo unit of sample per nucleotide material in each peak. Several HPLC methods have been reported for analyses of various nucleosides in tRNAs and nucleotide pools in tissues (IS). Van Boom and De Rooy (9) reported an HPLC method for sequencing oligonucleotides by estimating the amounts of 5'-mononucleotides and nucleosides sequentially liberated from the 3'-ends of the oligonucleotides by the action of snake venom phosphodiesterase. However, there have been no papers in which HPLC has been successfully applied for the determination of the nucleotide sequence of an RNA. Conditions of HPLC on columns of AS