[27] Controlled partial hydrolysis of RNA

Abstract

This chapter discusses the parameters that influence the rate and selectivity of chemical hydrolysis of RNA. Procedures are given for controlled hydrolysis to oligonucleotides. Acid and base catalyzed hydrolyses of RNA in the pH range 1-13 proceed via the attack of the 2' oxygen on the 3' phosphorus to form a 2',3' cyclic phosphate. This cyclic diester is then hydrolyzed to form mixed 2' and 3' phosphate-ended chains. In the pH range 2-11, the cyclic phosphate formation and its hydrolysis show similar pH dependence, but at pH 12-13, where the 2' OH is apparently completely ionized, cyclic ester formation is independent of pH while hydrolysis of the cyclic ester still increases as the pH is raised above 12. Thus, less cyclic ester accumulates during hydrolysis in strongly basic solution. Base-catalyzed hydrolysis of RNA is a two-step reaction. First the chain is cleaved by attack of the diester phosphate by the 2' hydroxyl to displace the 5' esterified portion of the chain and leave a 2',3' cyclic phosphate on the chain terminus. The hydrolytic opening of this cyclic phosphate depends on temperature, pH, catalyst, and water activity differently than does the reaction, which formed the cyclic ester. Thus, one may choose conditions, which favor the production of oligonucleotides with cyclic phosphate ends or with phosphomonoester ends.