Mapping adenines, guanines, and pyrimidines in RNA

TLDR

Partial nuclease digestion of terminally labeled RNA, using RNase T1 to cleave at guanines and RNase U2 at adenines, combined with alkaline hydrolysis, enables mapping of adenine, guanine, and pyrimidine positions along the molecule. The cleavage products are separated by size on a polyacrylamide gel and visualized by autoradiography, revealing the sequence up to 100 nucleotides from the 3′ end, though uracil and cytosine remain indistinguishable. These methods constitute a sequencing approach that was successfully applied to yeast 5.8S ribosomal RNA.

Abstract

The positions of adenines, guanines, and pyrimidines can be determined by partial nuclease digestion of a terminally labeles RNA molecule. In urea, at elevated temperatures, RNase T1 generates a pattern reflecting cleavage at guanines while RNase U2 cleaves only at adenine. A limited alkaline hydrolysis provides a continuum of fragments derived from breaks at every phosphodiester bond. The reaction products are electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel. An autoradiograph of the gel displays the sequence up to 100 nucleotides from the end of the molecule, although uracil cannot as yet be distinguished from cytosine. These techniques form the basis of an RNA sequencing method and are demonstrated on yeast 5.8S ribosomal RNA.