[3H]Borohydride: A Versatile Reagent for the Analysis of tRNA—Methods and Applications

Abstract

This review serves two purpose: (1) to outline ³H derivative methods for base composition and sequence analysis of nonradioactive RNA developed in our laboratory over the past 10 years and (2) to summarize results obtained in our laboratory by the application of these methods.

The first experiments designed to incorporate radioactive label into the end groups of nucleic acids by chemical means were reported 15–20 years ago by Khorana and his co-workers (Khorana 1959; Ralph et al. 1963; RajBhandary et al. 1964), but the principles enunciated by these workers have only recently found wide application in structural studies on nucleic acids. Thus far, the reagent most often used for labeling RNA 3′ ends has been [³H]borohydride. The labeling reaction entails the oxidation of the vicinal hydroxyl groups of the ribose moiety of nucleosides or nucleotides with sodium metaperiodate (NaIO₄), followed by the reduction of the resulting dialdehydes to diols:

In contrast to enzymatic ³²P labeling by the [γ-³²P]ATP-polynucleotide kinase reaction (see RajBhandary, this volume), ³H-label incorporation into the dialdehydes according to Scheme 1 is stoichiometric if the reduction is performed with excess borotritide (Randerath et al. 1972Randerath et al. 1979a). This has to be regarded as a major advantage of the ³H-labeling method, because the reaction is thus suitable for accurate quantitative determinations, e.g., of the base composition of polyribonucleotides or the molar ratios of oligoribonucleotides in enzymatic digests of RNA. Other advantages of the ³H-derivative methods include the availability of reproducible separation methods of high resolving power for...