title: A rapid and sensitive method for kinetic study and activity assay of DNase I in vitro based on a GO-quenched hairpin probe.
abstract: As a waste-management endonuclease, DNase I has been suggested to be one of the deoxyribonucleases responsible for DNA fragmentation during apoptosis. We report here an alternative fluorescence method for DNase I assay with high accuracy and sensitivity by applying a DNA/GO (graphene oxide) probe. The method with a detection limit of 1 U mL(-1) was then applied to investigate the effects of external factors including antibiotics and heavy metal ions on DNase I. The results demonstrated that gentamicin sulfate was a strong inhibitor with an IC50 value of 0.57 ± 0.12 mM. The investigated heavy metal ions showed an inhibitory effect on DNase I activity in a concentration dependent manner with IC50 values of 0.04 μg/mL (Hg(2+)), 0.10 μg/mL (Pb(2+)), 1.35 μg/mL (Cd(2+)), 1.20 μg/mL (As(2+)), and 1.80 μg/mL (Cu(2+)). Finally, the new method was applied to detect DNase levels in complicated tumor tissue and cell samples and the results showed that DNase levels increased in tumor tissues compared with that of adjacent tissue. From the above results, we conclude that the method can be widely used for high - throughput assay of DNase I in biological samples as well as drug screening in vitro. Graphical Abstract The schematic of real-time monitoring of DNase I using GO - quenched hairpin probe as the substrate. The process of nucleotide digestion catalyzed by DNase I produces short fragments of hairpin probe and accordingly causes a significant increase in fluorescence. At first, GO can absorb the hairpin probes and quenched their fluorescence. When there is DNase I, the DNase can cleave the double strands of DNA. Fluorescence is restored due to the significantly weaker binding ability of small DNA fragments to GO compared with long DNA fragments. So, we can detect the increase in fluorescence to study the activity of DNase.
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, 1st Denggao Road, Yuelu district, Changsha, Hunan, 410082, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, 1st Denggao Road, Yuelu district, Changsha, Hunan, 410082, China.
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Xie
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, 1st Denggao Road, Yuelu district, Changsha, Hunan, 410082, China. sw_tcy@hnu.edu.cn.
authors.forename: Chunyi
authors.initials: C
authors.lastname: Tong
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, 1st Denggao Road, Yuelu district, Changsha, Hunan, 410082, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Peng
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, 1st Denggao Road, Yuelu district, Changsha, Hunan, 410082, China.
authors.forename: Changhui
authors.initials: C
authors.lastname: Xiao
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, 1st Denggao Road, Yuelu district, Changsha, Hunan, 410082, China.
authors.forename: Xuanming
authors.initials: X
authors.lastname: Liu
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, 1st Denggao Road, Yuelu district, Changsha, Hunan, 410082, China.
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, 1st Denggao Road, Yuelu district, Changsha, Hunan, 410082, China. bioliu@hnu.edu.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism*; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D007700:Kinetics; D057230:Limit of Detection; D015335:Molecular Probes; D009369:Neoplasms / Q000201:enzymology; D013050:Spectrometry, Fluorescence
chemical_list: D000900:Anti-Bacterial Agents; D015335:Molecular Probes; D003850:Deoxyribonuclease I
keywords: DNase I; Graphene oxide; Hairpin probe; Real time; Tumor