Influence of Helix Length on Cleavage Efficiency of Hammerhead Ribozymes

Abstract

The cleavage rates of RNA substrates by trans-acting, hammerhead ribozymes are controlled by interactions between helices I and II. The interactions are affected by the relative lengths of these two double helices and by unpaired nucleotides protruding beyond helix I, either in the substrate or the ribozyme strand. Maximum cleavage rates are observed for ribozyme–substrate complexes with three or more base pairs in helix II and six or less base pairs in helix I. However, for these helix combinations, rates fall sharply with unpaired nucleotides at the end of helix I. Cleavage rates by ribozymes with one or two base pairs in helix II increase as helix I is lengthened, and are unaffected by unpaired nucleotides on the end. Since miniribozymes, with one base pair in helix II, efficiently cleave long RNA transcripts under physiological conditions, they represent the optimal design for the simple hammerheads for application in vivo.