Novel High-Throughput Deoxyribonuclease 1 Assay

Abstract

Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z’ ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.