The 10–23 DNAzyme is capable of cleaving RNA with high sequence specificity at sites that contain purine-pyrimidine (R-Y) junctions. Although they are abundant in mRNA, many of these potentially cleavable junctions are protected from DNAzyme activity by secondary structure. To optimise the process of target-site selection in long RNA substrates, a multiplex assay was developed for simultaneous comparative analysis of 50 or more different DNAzymes in one reaction. Using this approach, the efficiency of 80 DNAzyme sites within the E6 component of a full-length HPV16 E6/E7 transcript was examined. The activity of molecules selected in this system was then compared in a conventional assay with DNAzymes of intermediate and low performance. This confirmed the results observed in the multiplex reactions, with 10% of DNAzymes inducing substantial cleavage of the long transcript. These DNAzyme-sensitive regions are potentially accessible to other RNA directed agents such as ribozymes or antisense oligonucleotides. Therefore, in addition to finding the most effective DNAzymes for a particular target mRNA, this method may also be applicable to locating accessible sites for other nucleic acid-based gene suppression strategies.
