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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 Mn(2+). 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.

journal_name

Acta Pharmacol Sin

journal_title

Acta pharmacologica Sinica

authors

He HQ,Ma XH,Liu B,Zhang XY,Chen WZ,Wang CX,Cheng SH

doi

10.1111/j.1745-7254.2007.00561.x

subject

Has Abstract

pub_date

2007-06-01 00:00:00

pages

811-7

issue

eissn

1671-4083

issn

1745-7254

journal_volume

pub_type

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