3 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)223-230
Number of pages8
JournalAnalytical Biochemistry
Volume396
Issue number2
DOIs
StatePublished - Jan 15 2010

Fingerprint

Integrases
Assays
DNA
Antiviral Agents
Small Molecule Libraries
Denaturation
Fluorophores
Transition Temperature
Endonucleases
Viral DNA
Substrates
p31 integrase protein, Human immunodeficiency virus 1
Oligonucleotides
Melting point
Real-Time Polymerase Chain Reaction
Screening
Hot Temperature
Fluorescence
Gels
Throughput

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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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.",
author = "Malgorzata Sudol and Melissa Tran and Nowak, {Matthew G.} and John Flanagan and Gavin Robertson and Michael Katzman",
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doi = "10.1016/j.ab.2009.09.012",
language = "English (US)",
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A nonradioactive plate-based assay for stimulators of nonspecific DNA nicking by HIV-1 integrase and other nucleases. / Sudol, Malgorzata; Tran, Melissa; Nowak, Matthew G.; Flanagan, John; Robertson, Gavin; Katzman, Michael.

In: Analytical Biochemistry, Vol. 396, No. 2, 15.01.2010, p. 223-230.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A nonradioactive plate-based assay for stimulators of nonspecific DNA nicking by HIV-1 integrase and other nucleases

AU - Sudol, Malgorzata

AU - Tran, Melissa

AU - Nowak, Matthew G.

AU - Flanagan, John

AU - Robertson, Gavin

AU - Katzman, Michael

PY - 2010/1/15

Y1 - 2010/1/15

N2 - 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.

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