Characterization of endonucleolytic activity of HIV-1 integrase using a fluorogenic substrate.
Retroviruses require viral DNA to be synthesized by reverse transcription in the cytoplasm followed by integration of the resulting viral DNA into the host chromosome in the nucleus. Reverse transcription and integration, essential steps in the life cycle of retroviruses, are possible targets in the development of antiviral reagents. One attractive target is the integrase protein, a product of the retroviral pol gene which is solely responsible for the retroviral integration process through cutting and joining reactions. When screening for massive numbers of antiviral agents, a rapid and precise assay is ideal. We report the application of fluorescence resonance energy transfer (FRET) with fluorescein and eosin as the energy transfer pair to characterize HIV-IN-mediated DNA cleavage reactions. Past concerns with applications of FRET to DNA were due to interactions of the fluorophore with the DNA, resulting in quenched fluorescence. However, in this study these concerns have been resolved with the use of a nucleotide analog with a 12-carbon linker arm, 5-amino (12)-2'-deoxyuridine beta-cyanoethyl phosphoramidite. Steady-state fluorescence studies show that cleavage of the fluorogenic substrate by integrase results in enhancement of quenched donor fluorescence intensity. The fluorescence assay was confirmed by autoradiographic analysis of the cleavage reaction with radiolabeled fluorogenic substrate. This fluorescence assay will facilitate both detailed kinetic studies and the rapid screening of novel integrase inhibitors.
Lee
S P
SP
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.
Censullo
M L
ML
Kim
H G
HG
Knutson
J R
JR
Han
M K
MK
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D003857:Deoxyuridine / Q000031:analogs & derivatives; D004706:Endodeoxyribonucleases / Q000032:analysis*; D004735:Energy Transfer; D004801:Eosine Yellowish-(YS) / Q000737:chemistry*; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes; D019427:HIV Integrase; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence
C095883:5-amino (12)-2'-deoxyuridine beta-cyanoethyl phosphoramidite; D005452:Fluoresceins; D005456:Fluorescent Dyes; D004247:DNA; D019427:HIV Integrase; D004706:Endodeoxyribonucleases; C058608:endonuclease-integrase; D004801:Eosine Yellowish-(YS); D019793:Fluorescein; D003857:Deoxyuridine