EFFECTS OF HELICAL STRUCTURES FORMED BY THE BINDING ARMS OF DNAZYMES AND THEIR SUBSTRATES ON CATALYTIC ACTIVITY

As a part of our efforts to clarify structure-function relationships i n reactions catalyzed by deoxyribozymes (DNAzymes), which were recentl y selected in vitro, we synthesized various chimeras and analyzed the kinetics of the corresponding cleavage reactions. We focused on the bi nding arms and generated helices composed of binding arms and substrat es that consisted of RNA and RNA, of RNA and DNA or of DNA and DNA, As expected for the rate limiting chemical cleavage step in reactions ca talyzed by DNAzymes, a linear relationship between log(k(cat)) and pH was observed, In all cases examined, introduction of DNA into the bind ing helix enhanced the rate of chemical cleavage. Comparison of CD spe ctra of DNAzyme substrate complexes suggested that higher levels of B- form-like helix were associated with higher rates of cleavage of the s ubstrate within the complex. To our surprise, the enhancement of catal ytic activity that followed introduction of DNA into the binding helix (enhancement by the presence of more B-form-like helix) was very simi lar to that observed in the case of the hammerhead ribozymes that we h ad investigated previously. These data, together with other observatio ns, strongly suggest that the reaction mechanism of metal-ion-dependen t DNAzymes is almost identical to that of hammerhead ribozymes.