title: An end-point method based on graphene oxide for RNase H analysis and inhibitors screening.
abstract: As a highly conserved damage repair protein, RNase H can hydrolysis DNA-RNA heteroduplex endonucleolytically and cleave RNA-DNA junctions as well. In this study, we have developed an accurate and sensitive RNase H assay based on fluorophore-labeled chimeric substrate hydrolysis and the differential affinity of graphene oxide on RNA strand with different length. This end-point measurement method can detect RNase H in a range of 0.01 to 1 units /mL with a detection limit of 5.0×10-3units/ mL under optimal conditions. We demonstrate the utility of the assay by screening antibiotics, resulting in the identification of gentamycin, streptomycin and kanamycin as inhibitors with IC50of 60±5µM, 70±8µM and 300±20µM, respectively. Furthermore, the assay was reliably used to detect RNase H in complicated biosamples and found that RNase H activity in tumor cells was inhibited by gentamycin and streptomycin sulfate in a concentration-dependent manner. The average level of RNase H in serums of HBV infection group was similar to that of control group. In summary, the assay provides an alternative tool for biochemical analysis for this enzyme and indicates the feasibility of high throughput screening inhibitors of RNase H in vitro and in vivo.
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha 410082, China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Zhao
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha 410082, China.
authors.forename: Jialong
authors.initials: J
authors.lastname: Fan
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha 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, Changsha 410082, China.
authors.forename: Lijian
authors.initials: L
authors.lastname: Zhao
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha 410082, China. Electronic address: sw_tcy@hnu.edu.cn.
authors.forename: Chunyi
authors.initials: C
authors.lastname: Tong
authors.affiliation: School of Pharmaceutical Science, Hunan University of Chinese Medicine, Changsha 410208, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha 410082, China. Electronic address: binliu2001@hotmail.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
mesh_terms: D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry; D005839:Gentamicins / Q000627:therapeutic use; D006108:Graphite / Q000737:chemistry*; D006509:Hepatitis B / Q000097:blood* / Q000188:drug therapy; D006801:Humans; D007612:Kanamycin / Q000627:therapeutic use; D010087:Oxides / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D016914:Ribonuclease H / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D013307:Streptomycin / Q000627:therapeutic use
chemical_list: D005839:Gentamicins; D010087:Oxides; D007612:Kanamycin; D012313:RNA; D006108:Graphite; D004247:DNA; D016914:Ribonuclease H; D013307:Streptomycin
keywords: Graphene Oxide; HBV; Inhibitor screening; RNase H