Chemical modification of ribonucleic acid. A direct study by carbon-13 nuclear magnetic resonance spectroscopy

Direct study of the methylation of ribonucleic acid with methyl methanesulfonate by carbon-13 nuclear magnetic resonance spectroscopy has demonstrated the usefulness of this method in studying the chemical modification of biomacro-molecules and the interaction between nucleic acids and biologically active agents. This direct stable isotope method eliminated all tedious and questionable degradation processes for determining the reactive sites and the product distributions. Six methylated products, 7-methylguanosine, 1-methyladenosine, 3-methylcytidine, 1-methylguanosine, 3-methyluridine, and methyl phosphodiester, were identified by comparison with many model compounds and careful examination of spin-spin coupling and spin-lattice relaxation time. An extensive study of the interaction of phosphate buffer with methyl methansulfonate accounted for the sharp difference in the 13C spectra of the methylated RNA isolated from the reactions controlled by a pH-stat and phosphate buffer, respectively. The 13C-enriched agent significantly enhances the specificity and sensitivity of the method and provides better quantitative results.