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Simple and convenient G-quadruplex-based turn-on fluorescence assay for 3' ? 5' exonuclease activity.

ABSTRACT: A selective, oligonucleotide-based, label-free, turn-on fluorescence detection method for 3' ? 5' exonuclease activity has been developed using crystal violet as a G-quadruplex-binding probe. The assay is highly simple and rapid, does not require the use of gel-based equipment or radioisotopic labeling, and is amenable to high-throughput and real-time detection. A proof-of-concept of this assay has been demonstrated for prokaryotic Exonuclease III (ExoIII) and human TREX1.

SUBMITTER: Leung CH

PROVIDER: S-EPMC3021625 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Simple and convenient G-quadruplex-based turn-on fluorescence assay for 3' → 5' exonuclease activity.

Leung Chung-Hang CH Chan Daniel Shiu-Hin DS Man Bradley Yat-Wah BY Wang Chuan-Jen CJ Lam Wing W Cheng Yung-Chi YC Fong Wang-Fun WF Hsiao Wen-Luan Wendy WL Ma Dik-Lung DL

Analytical chemistry 20101129 2

A selective, oligonucleotide-based, label-free, turn-on fluorescence detection method for 3' → 5' exonuclease activity has been developed using crystal violet as a G-quadruplex-binding probe. The assay is highly simple and rapid, does not require the use of gel-based equipment or radioisotopic labeling, and is amenable to high-throughput and real-time detection. A proof-of-concept of this assay has been demonstrated for prokaryotic Exonuclease III (ExoIII) and human TREX1. ...[more]

PMID: 21114271

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Project description:BackgroundThe assembly and release of human immunodeficiency virus (HIV) particles from infected cells represent attractive, but not yet exploited targets for antiretroviral therapy. The availability of simple methods to measure the efficiency of these replication steps in tissue culture would facilitate the identification of host factors essential for these processes as well as the screening for lead compounds acting as specific inhibitors of particle formation. We describe here the development of a rapid cell based assay for quantification of human immunodeficiency virus type 1 (HIV-1) particle assembly and/or release.ResultsUsing a fluorescently labelled HIV-derivative, which carries an eYFP domain within the main viral structural protein Gag in the complete viral protein context, the release of virus like particles could be monitored by directly measuring the fluorescence intensity of the tissue culture supernatant. Intracellular Gag was quantitated in parallel by direct fluorescence analysis of cell lysates, allowing us to normalize for Gag expression efficiency. The assay was validated by comparison with p24 capsid ELISA measurements, a standard method for quantifying HIV-1 particles. Optimization of conditions allowed the robust detection of particle amounts corresponding to 50 ng p24/ml in medium by fluorescence spectroscopy. Further adaptation to a multi-well format rendered the assay suitable for medium or high throughput screening of siRNA libraries to identify host cell factors involved in late stages of HIV replication, as well as for random screening approaches to search for potential inhibitors of HIV-1 assembly or release.ConclusionsThe fast and simple fluorescence based quantification of HIV particle release yielded reproducible results which were comparable to the well established ELISA measurements, while in addition allowing the parallel determination of intracellular Gag expression. The protocols described here can be used for screening of siRNA libraries or chemical compounds, respectively, for inhibition of HIV in a 96-well format.

Dataset Information

Simple and convenient G-quadruplex-based turn-on fluorescence assay for 3' ? 5' exonuclease activity.

Publications

Simple and convenient G-quadruplex-based turn-on fluorescence assay for 3' → 5' exonuclease activity.

Similar Datasets

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