title: A nonradioactive plate-based assay for stimulators of nonspecific DNA nicking by HIV-1 integrase and other nucleases.
abstract: Retroviral integrase enzymes have a nonspecific endonuclease activity that is stimulated by certain compounds, suggesting that integrase could be manipulated to damage viral DNA. To identify integrase stimulator (IS) compounds as potential antiviral agents, we have developed a nonradioactive assay that is suitable for high-throughput screening. The assay uses a 49-mer oligonucleotide that is 5'-labeled with a fluorophore, 3'-tagged with a quencher, and designed to form a hairpin that mimics radioactive double-stranded substrates in gel-based nicking assays. Reactions in 384-well plates are analyzed on a real-time PCR machine after a single heat denaturation and subsequent cooling to a point between the melting temperatures of unnicked substrate and nicked products (no cycling is required). Under these conditions, unnicked DNA reforms the hairpin and quenches fluorescence, whereas completely nicked DNA yields a large signal. The assay was linear with time, stimulator concentration, and amount of integrase, and 20% concentrations of the solvent used for many chemical libraries did not interfere with the assay. The assay had an excellent Z' factor, and it reliably detected known IS compounds. This assay, which is adaptable to other nonspecific nucleases, will be useful for identifying additional IS compounds to develop the novel antiviral strategy of stimulating integrase to destroy retroviral DNA.
authors.affiliation: Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033, USA.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Sudol
authors.forename: Melissa
authors.initials: M
authors.lastname: Tran
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Nowak
authors.forename: John M
authors.initials: JM
authors.lastname: Flanagan
authors.forename: Gavin P
authors.initials: GP
authors.lastname: Robertson
authors.forename: Michael
authors.initials: M
authors.lastname: Katzman
mesh_terms: D004247:DNA / Q000032:analysis*; D053904:DNA Breaks, Single-Stranded; D003851:Deoxyribonucleases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D019427:HIV Integrase / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D004247:DNA; D019427:HIV Integrase; D003851:Deoxyribonucleases; C514801:p31 integrase protein, Human immunodeficiency virus 1