High Performance Liquid Chromatography of Nucleosides in RNA and DNA

Abstract

Reversed-phase high performance liquid chromatography has been developed and used effectively as a research tool for the quantitative analysis of major and modified nucleosides present in RNAs, DNAs, and physiological fluids. Gehrke et al. (5, 6, 24, 28) have shown that RP-HPLC is especially well suited for the analysis of the array of modified nucleosides found in tRNA, as more than forty nucleosides can be resolved and quantitatively determined in a single analysis. Coupled with our rapid, quantitative and straightforward enzymatic hydrolysis protocol, RP-HPLC compositional analyses can be directly performed on microgram quantities of either unfractionated or isoaccepting tRNAs. This method is applicable to the comparison of nucleoside compositions of tRNAs from parental and mutant organisms. In addition, Gehrke and Kuo (31, 33) have developed a highly precise RP-HPLC method for the analysis of the methylated nucleoside present in DNA, 5-methyldeoxycytidine, which has been used in collaborative research to measure differences in the extent of methylation of DNA from a range of cell and tissue types and DNA sequences. The deoxyribonucleoside 3′-and 5′-monophosphates, including pm⁵dC, are also well resolved by RP-HPLC, and this separation technique should prove of value in studies on sequence methylation in DNA. RP-HPLC analysis preceded by boronate gel selective isolation of ribonucleosides gives an effective technique for the analysis of ribonucleosides in physiological fluids, and a number of publications show that urinary nucleoside levels can serve as useful indicators of neoplastic disease status.