Directed cleavage of ribopolynucleotides with nucleases restricted by multiple modification of substrate

Abstract

Simultaneous exhaustive modification of cytidine and uridine residues of rRNA with methoxyamine and sodium metabisulfite renders adjacent phosphodiester bonds resistant to pancreatic and T 2 ribonucleases. Another method of T 2 RNAase restriction is modification of cytidine with methoxyaminebisulfite followed by modification of guanosine residues with β-ethoxy-α-ketobutyraldehyde. Mild alkaline treatment leads to demodification of uridine and guanosine residues leaving intact modified cytidine residues, thus providing a means of stepwise, directed cleavage of the polynucleotide. The series of combined cleavage procedures and methods of isolation of oligo(C), oligo(G) and oligopyrimidine tracts, as well as the procedure of selective cleavage at uridine residues elaborated in the course of the present studies may serve as a basis for more rational procedures of RNA sequencing.