Ribonucleoside analysis by reversed-phase high-performance liquid chromatography

Abstract

Over the past fifteen years we have developed and refined the analytical chromatographic methodologies using reversed-phase high-performance liquid chromatography and UV—photodiode array detection (RPLC—UV) for the detection and measurement of the major and modified nucleosides in nucleic acids and biological fluids. RPLC—UV nucleoside analysis as it has now evolved is a powerful new research tool to aid investigators in the fields of biochemical and biomedical research. This RPLC—UV nucleoside method can resolve more than 65 nucleosides in a single analysis with “run-to-run” peak retention variations of less than 1%. A complete nucleoside composition can be obtained from as little as 0.5 μg RNA. Identification and confirmation of nucleosides can be made from the highly reproducible retention times and from the characteristic UV spectrum from a few picomoles ( ca. 1 ng) of nucleoside. In this paper we introduce standard RPLC—UV methodologies for the analysis of nucleosides and nucleoside composition of RNAs. The chromatographic protocols and standard nucleoside columns are presented and the essential requirements necessary in the HPLC instrumentation are described. Three optimized RPLC systems were developed, each with particular emphasis placed on resolution, speed, or sensitivity. In addition, three unfractionated tRNAs were selected as sources of reference nucleosides are for assessment of the performance of the chromatography. From these tRNAs, a large array of nucleosides were characterized which are used in standardization and calibration of the method. Also discussed is the use of a diode-array detector for enhancement of the reliability of nucleoside identification and accuracy of measurement. An extended enzymatic hydrolysis protocol for the liberation of exotically modified nucleosides in tRNAs is also described. Chromatographic retention times and UV spectra for a large number of ribonucleosides are tabulated. The RPLC—UV ribonucleoside analytical protocols are capable of quantifying 31 nucleosides. Approximately 1 μg of an isoaccepting tRNA, or 20 μg of unfractioned tRNA are needed for quantitative analysis. With this amount of tRNA, the percent relative error of measurement of the four major nucleosides is less than 2%, and for the modified nucleosides about 5%. As little as 0.2 μg of pure isoaccepting tRNA can be analyzed, but at the expense of precision as at this low sample size a 20–30% relative error for modified nucleosides is to be expected. For quantitation of the modified nucleosides in rRNA, which contains much less modification than tRNAs, 10–100 μg of sample are needed per injection. This amount is within the loading capacity of a regular analytical column (25 cm × 4.6 mm silica based C 18 column). However, with this quantity injected, caution is required to ensure that the response for the four major nucleosides is within the linear range of the detector and data reduction system. Quantitative data from the analysis of 16S and 23S rRNA are given. Examples are presented of some unique and interesting applications of this nucleoside methodology to biochemical and biomedical investigations.