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

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 IC50 values 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. © 2007 CPS and SIMM.