Construction of new ribozymes requiring short regulator oligonucleotides as a cofactor

A hairpin loop and an oligonucleotide bound to the loop form one-half of th e pseudoknot structure. We have designed an allosteric hammerhead ribozyme, which is activated by the introduction of this motif by using a short comp lementary oligonucleotide as a cofactor. Stem II. of the hammerhead ribozym e was substituted with a non-self-complementary loop sequence (loop II) to abolish the cleavage activity. The new ribozyme had almost no cleavage acti vity of the target RNA. However, it exhibited the cleavage activity in the presence of a cofactor oligoribonucleotide, which is complementary to loop II of the ribozyme. The activity is assumed to be derived from the formatio n of a pseudo-stem structure between the cofactor oligonucleotide and loop II. The structure including the loop may be similar to the pseudo-half-knot structure. The activation efficiencies of the cofactor oligonucleotides we re decreased as the lengths of the oligonucleotides increased, and the ribo zyme with a longer loop II was more active than that with a short loop II. Oligoribonucleotides with 3'-dangling purine bases sen ed as efficient cofa ctors of the ribozyme, and a 2'-O-methyloligonucleotide enhanced the cleava ge activity of the ribozyme most efficiently, by as much as about 750-fold as compared with that in the absence of the oligonucleotide. Cofactor oligo nucleotides with a cytidine base at the 3'-end also activated a ribozyme wi th the G10.1 G11.1 mutation, which eliminates the cleavage activity in the wildtype. The binding sites of the oligonucleotide were identified by photo crosslinking experiments and were found to be the predicted sites in the lo op. This is the first report of a design aimed at positively controlling th e activity of ribozymes by employing a structural motif. This method can be applied to control the activities of other functional RNAs with hairpin lo ops. (C) 2000 Academic Press.