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


CONCLUSIONS
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.
He
Hong-Qiu
HQ
College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100022, China.
Ma
Xiao-Hui
XH
Liu
Bin
B
Zhang
Xiao-Yi
XY
Chen
Wei-Zu
WZ
Wang
Cun-Xin
CX
Cheng
Shao-Hui
SH
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
D005456:Fluorescent Dyes; D019427:HIV Integrase