Journal
JOURNAL OF BIOMOLECULAR SCREENING
Volume 20, Issue 2, Pages 202-211Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057114555828
Keywords
DNase I; high-throughput assay; near-infrared fluorescence; cisplatin; kidney
Funding
- National Institutes of Health [R01 DK078908, R01 CA140409, P20 RR016460-11, P20 GM103429-11]
- US Department of Defense Telemedicine & Advanced Technology Research Center (DOD TATRC)
- US Department of Veterans Affairs (VA) Merit Review grant [I01 BX000690]
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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.
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