Modified 5′-nucleotides: A general procedure for their isolation from phosphodiesterase hydrolysates of transfer RNA

Abstract

A procedure is described for the isolation and purification of a wide variety of modified 5′-nucleotides from a single, large-scale phosphodiesterase hydrolysate of transfer RNA. The procedure involves the following: (i) initial resolution of hydrolysis products into six major groups by chromatography on a large column of DEAE-cellulose; (ii) further resolution of each of these groups by chromatography on smaller columns of DEAE-cellulose or Dowex-1; (iii) preparative paper chromatography of the resulting subfractions, in order to separate and purify individual 5′-nucleotides. Use of formic acid – formate buffers during column chromatography facilitates desalting and (or) recovery of fractions, while the use of volatile organic solvents during paper chromatography largely eliminates the necessity of a desalting step during the final recovery of the individual, purified compounds.With this procedure, we have isolated (as the corresponding 5′-nucleotides) virtually all of the modified nucleosides which have been identified to date in wheat embryo tRNA. These include m1A, m6A, Am, c-oi6A, tc6A, m6tc6A, I, m1l, m3C, m5C, Cm, ac4C, m1G, m2G, m22G, Gm, m7G, Q, m5U, Um, nbt3U, ncm5U, Ψ, Ψm, and h2U (symbols are in accord with the recommendations of the IUPAC–IUB Commission on Biochemical Nomenclature; see Dunn, D. B. &Hall, R. H. (1975) in Handbook ofBiochemistry and Molecular Biology (Fasman, G. D., ed.), 3rd ed., vol. 1, pp. 65–215, CRC Press, Cleveland, OH). Mobilities for 24 modified 5′-nucleotides in four paper chromatographic systems have been determined.The procedure described is generally applicable to other types of RNA and could undoubtedly be adapted to prepare modified 5′-nucleotides other than those which can be recovered from phosphodiesterase hydrolysates of wheat embryo tRNA.