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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.

SUBMITTER: Jang DS 

PROVIDER: S-EPMC4564109 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Novel high-throughput deoxyribonuclease 1 assay.

Jang Dae Song DS   Penthala Narsimha R NR   Apostolov Eugene O EO   Wang Xiaoying X   Fahmi Tariq T   Crooks Peter A PA   Basnakian Alexei G AG  

Journal of biomolecular screening 20141017 2


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 intens  ...[more]

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