High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction
High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3’-processing reaction作者机构：College of Life Science and Bioengineering Beijing University of Technology Beijing China Department of Pharmacentical and Biomedical Sciences University of Georgia Athens USA
出 版 物：《Acta Pharmacologica Sinica》 (中国药理学报(英文版))
年 卷 期：2007年第28卷第6期
页 面：811-817页
核心收录：
学科分类：1007[医学-药学(可授医学、理学学位)] 1006[医学-中西医结合] 100706[医学-药理学] 100602[医学-中西医结合临床] 10[医学]
基 金：the National Natural Science Foundation of China(№ 30500429 and 30670497) the Beijing Natural Science Foundation(№ 5072002)
主 题：HIV integrase 3’-processing molecular beacons
摘 要：Aim:To develop a high-throughput real-time assay based on molecular beaconsto monitor the integrase 3’-processing reaction in vitro and apply it to ***:The recombinant human immunodeficiency virus (HIV)-1integrase (IN) is incubated with a 38 mer oligonucleotide substrate,a sequenceidentical to the U5 end of HIV-1 long terminal repeats (LTR).Based on the fluores-cence properties of molecular beacons,the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5’ end and a quencher at the 3’ *** cleaves the terminal 3’-dinucleotide containing the quencher,resulting in anincrease in fluorescence which can be monitored on a *** this assay,tests were performed to investigate the effects of substrates,enzyme and the metal ion concentrations on the IN activity and optimal param-eters were ***,2 IN inhibitors were employed to test the perfor-mance of this assay in antiviral compound ***:The fluorescentintensity of the reaction mixture varies linearly with time and is proportional to thevelocity of the 3’-processing *** were performed and the results showedthat the optimal rate was obtained for a reaction mixture containing 50 mg/L recom-binant HIV-1 IN,400 nmol/L substrate,and 10 mmol/L Mn2+.The IN 3’-processingreaction under the optimal conditions showed a more than 18-fold increase in thefluorescence intensity compared to the enzyme-free *** IC50values ofthe IN inhibitors obtained in our assay were similar to the values obtained from aradiolabeled substrate ***:Our results demonstrated that this is afast,reliable,and sensitive method to monitor HIV IN 3’-processing reaction andthat it can be used for inhibitor screening.
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