title: An ultrasensitive fluorescence method suitable for quantitative analysis of mung bean nuclease and inhibitor screening in vitro and vivo.
abstract: Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease 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: 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: 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. Electronic address: 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, Changsha, 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, 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: 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, Changsha, 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, Changsha, 410082, China. Electronic address: binliu2001@hotmail.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D004277:DNA, Single-Stranded / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D004791:Enzyme Inhibitors / Q000378:metabolism; D007887:Fabaceae / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D005453:Fluorescence; D006108:Graphite / Q000737:chemistry; D057230:Limit of Detection; D010087:Oxides / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; C526101:Mung Bean Nuclease; D010087:Oxides; D010940:Plant Proteins; D006108:Graphite; D015719:Single-Strand Specific DNA and RNA Endonucleases
keywords: Fluorescence; Graphene oxide; Mung bean nuclease; Quantitative analysis