Original Article

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

Authors: Hong-qiu He, Xiao-hui Ma, Bin Liu, Xiao-yi Zhang, Wei-zu Chen, Cun-xin Wang, Shao-hui Cheng

Abstract

Aim: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening.
Methods: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluorescence 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' end. IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal parameters were obtained. Moreover, 2 IN inhibitors were employed to test the performance of this assay in antiviral compound screening.
Results: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recombinant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn2+. The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay.
Conclusion: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.
Keywords:

Download Citation

Cited times in Scopus