Novel high‐throughput DNase I assay

Abstract

DNase I is the most active and abundant endonuclease in the body, mediates toxic injury of kidney cells induced by cisplatin. This presents a major obstacle for cancer chemotherapy. However, neither inhibitors of DNase I nor high‐throughput screening methods for screening of high‐volume chemical libraries for DNase I inhibitors are available. To overcome this problem, we have developed a high‐throughput DNase I assay. The assay is based on the increase of fluorescence intensity when fluorophore‐labeled oligonucleotides are degraded by a DNase. It is optimized for a 96‐well plate format. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z′≥0.6), operationally simple, and has low intra‐ and inter‐assay variability. The assay was used to screen a chemical library and several potential DNase I inhibitors were identified. After comparison, one best inhibitor was selected and shown to protect against cisplatin‐induced kidney cell death in vitro. This assay will be suitable for identifying potential inhibitors of DNase I and other endonucleases. NIH R01 DK078908 and VA Merit Review grants to AGB.