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A method is described which allows a very efficient determination of the modified nucleosides of tRNA. The technique involves enzymatic degradation of the tRNA to nucleosides at pH 7.6 and their separation by two-dimensional thin-layer chromatography on cellulose-coated aluminum foils. Based on the analysis of two mammalian tRNAs it is shown that the technique is suitable for the determination of chemically unstable nucleosides as well as the ribose-methylated compounds. At least 36 of the 45 known modified nucleosides can be separated and quantitatively determined by the method described. This procedure is especially suitable for the estimation of the nucleoside composition of unlabeled tRNAs as well as for studying the post-transcriptional modifications of tRNA.
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