Target site selection for an RNA-cleaving catalytic DNA

A small catalytic DNA, known as the 10-23 DNA enzyme or deoxyribozyme, has been shown to efficiently hydrolyze RNA at purine-pyrimidine (R-Y) junction s in vitro. Although these potentially cleavable junctions are ubiquitous, they are often protected from deoxyribozyme activity by RNA secondary struc ture. We have developed a multiplex cleavage assay for screening the entire length of a target RNA molecule for deoxyribozyme cleavage sites that are efficient, both in terms of kinetics and accessibility. This strategy allow ed us to simultaneously compare the RNA cleaving activity of 80 deoxyribozy mes for a model target gene (HPV16 E6), and an additional 60 deoxyribozymes against the rat c-myc target. The human papilloma virus (HPV) target was u sed primarily to characterize the multiplex system and determine its validi ty. The c-myc target, coupled with a smooth muscle cell proliferation assay , allowed us to assess the relationship between in vitro cleavage efficienc y and c-myc gene suppression in cell culture. The multiplex reaction approa ch streamlines the process of revealing effective deoxyribozymes in a funct ional assay and provides accessibility data that may also be applicable to site selection for other hybridization-based agents.