Chemiluminescent Deoxyribozyme Sensors for DNA-Editing Enzymes

DNA-editing enzymes such as those in the APOBEC family of cytidine deaminases play important roles in both normal and pathogenic function, while engineered enzymes offer exciting new possibilities for genome editing. Despite their importance, widely used assays for DNA-editing enzymes are time-consuming and expensive. Here, we describe a new assay for DNA-editing enzymes in which the substrate in the reaction is a chemiluminescent deoxyribozyme called Supernova. Editing alters the sequence of Supernova, which results in a change in catalytic activity and light production. By analyzing a data set of Supernova variants previously identified by selection and high-throughput sequencing, it was possible to generate sensors with a wide range of specificities. Sensors were also developed for APOBEC3A, a cytidine deaminase which converts C to U in single-stranded DNA and RNA. These include a turn-off sensor that produces light 14-fold slower after incubation with recombinant APOBEC3A than in its absence, and a turn-on sensor that generates light 10-fold faster after incubation with APOBEC3A than in its absence. Assays that use these sensors are faster and less expensive than existing ones, and should be particularly useful for applications such as high-throughput screening.