HPLC based single-step kinetic assay to screen the activity of DNAzymes

DNAzymes are a new class of biosensors and pharmaceuticals whose mode-of-action is to cleave RNA. It is important to determine their cleavage rate during discovery and optimization as well as in quality control. We developed an automatable AEX-HPLC method to assess their cleavage rate in a single manual working step. The method obtains cleavage kinetics without substrate labeling and different DNAzymes can be tested simultaneously. We used this method to determine the two most active cleaving DNAzymes of our selection, DzTHM290 and DzTHM95, and evaluated their temperature dependency in comparison to a predetermined benchmark DNAzyme. The DNAzymes were designed to combat difficult-to-treat respiratory tract infections caused by mucoid P. aeruginosa. The mRNA of the target protein AlgD (GDP-mannose 6-dehydrogenase) was identified as the strongest candidate in blocking the alginate formation of mucoid P. aeruginosa isolates.