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 the pseudoknot structure. We have designed an allosteric hammerhead ribozyme, which is activated by the introduction of this motif by using a short complementary oligonucleotide as a cofactor. Stem II of the hammerhead ribozyme was substituted with a non-self-complementary loop sequence (loop II) to abolish the cleavage activity. The new ribozyme had almost no cleavage activity 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 formation 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 were decreased as the lengths of the oligonucleotides increased, and the ribozyme with a longer loop II was more active than that with a short loop II. Oligoribonucleotides with 3'-dangling purine bases served as efficient cofactors of the ribozyme, and a 2'-O-methyloligonucleotide enhanced the cleavage activity of the ribozyme most efficiently, by as much as about 750-fold as compared with that in the absence of the oligonucleotide. Cofactor oligonucleotides with a cytidine base at the 3'-end also activated a ribozyme with the G10.1.G11.1 mutation, which eliminates the cleavage activity in the wild-type. The binding sites of the oligonucleotide were identified by photo-crosslinking experiments and were found to be the predicted sites in the loop. This is the first report of a design aimed at positively controlling the activity of ribozymes by employing a structural motif. This method can be applied to control the activities of other functional RNAs with hairpin loops.

Induction of ribozyme activity by anti-ribozyme oligonucleotides.

A new type of hammerhead ribozyme, with cleavage activity enhanced by oligonucleotides, was constructed. Stem II of the ribozyme was substituted with a non complementary loop (loop II). The modified ribozyme exhibited negligible cleavage of a target RNA; however, it was converted to an active molecule in the presence of oligonucleotides which were complementary to loop II. The oligonucleotide compensated for the disabled stem II by binding with the ribozyme. The induction of the cleavage activity was sequence-specific and the oligonucleotides containing a purine base as the 3'-dangling end were able to induce the cleavage activity of the ribozyme most efficiently. A photo-crosslinking experiment proved that a pseudo-half-knot structure was formed in the active molecule. The cleavage of two kinds of substrate RNAs with different sequences was controlled by the corresponding ribozymes activated by specific oligonucleotides.

The beginning of Menière's disease.

Sudden deafness and low-tone hearing loss were treated by steroid therapy. Of the 106 cases of sudden deafness, 23 (21.7%) showed complete recovery, 36 (34.0%) remarkable improvement and 13 (12.3%) no change, while the results for the 56 cases of low-tone hearing loss were 32 (57.1%), 1 (1.8%), 16 (28.6%) and 7 (12.5%), respectively. Of all patients, 37 had onset of vertigo within 2 to 24 months after steroid therapy, and 17 patients, including 4 with horizontal type, 2 with deaf type and 11 with low-tone hearing loss, were diagnosed with Menière's disease. The final hearing gain for 4 cases of horizontal type and 2 cases of deaf type sudden deafness was very great. The 17 patients diagnosed with Menière's disease had hydrops of the inner ear.