Base analysis of ribopolynucleotides by chemical tritium labeling: A methodological study with model nucleosides and purified tRNA species

(1) A method for determining the major and minor base composition of ribopolynucleotides is described, which consists of the following steps: (a) Enzymic digestion of the ribopolynucleotide to a mixture of nucleosides. (b) Oxidation of the digest with periodate. (c) Reduction with tritiated borohydride to labeled nucleoside derivatives. (d) Two-dimensional thin-layer chromatography on cellulose. (e) Liquid scintillation counting. (2) The method is extremely sensitive: tritium-labeled digest derived from less than 1 μg of polynucleotide is required to evaluate the base composition, including most of the so-called minor or modified bases which are natural constituents of RNA. (3) The method has been studied by using both ribonucleosides and tRNA's of known structure as model compounds. (4) Conditions have been chosen for the overall procedure under which RNA constituents are preserved that are partially or completely destroyed under the usual conditions for alkaline hydrolysis of RNA. (5) In the course of this investigation, molar absorptivities were determined for dihydrouridine, 1-methylguanosine, and N2,N2-dimethylguanosine.