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A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands.

ABSTRACT: Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5' to 3' DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.

SUBMITTER: Mendoza O

PROVIDER: S-EPMC4477640 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands.

Mendoza Oscar O Gueddouda Nassima Meriem NM Boulé Jean-Baptiste JB Bourdoncle Anne A Mergny Jean-Louis JL

Nucleic acids research 20150312 11

Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these st ...[more]

PMID: 25765657

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Project description:BackgroundThe emergence and spread of drug resistance in Trichomonas vaginalis parasites has become an important concern in trichomoniasis treatment. Fast and reliable growth assessment is critical for validating in vitro drug susceptibility and high-throughput screening of newly developed drugs.MethodsModified media without yeast extract were evaluated for their ability to support the growth of T. vaginalis parasites. The potential of the nucleic acid-binding dye SYBR Green I for detecting T. vaginalis drug resistance was characterized, and seeding parasite concentration and incubation time were optimized. The fluorescence assay based on SYBR Green I was further validated in four T. vaginalis isolates with different susceptibilities to the antibiotics metronidazole, tinidazole, ornidazole and secnidazole, and compared with the traditional method that detects minimum lethal concentrations (MLCs).ResultsA modified medium consisting of RPMI 1640 and Tryptone Plus as replacements for yeast extract and tryptone, respectively, in traditional trypticase-yeast extract-maltose (TYM) medium exhibited similar performance as TYM medium in maintaining T. vaginalis growth, while it showed much lower background fluorescent signals. The T. vaginalis SYBR Green I-based fluorescence (TSF) drug assay was found to have to satisfy one of two conditions to demonstrate the 50% inhibitory concentration of metronidazole for the sensitive isolate TV-334: (i) a seeding density of 3 × 104 parasites/ml and an incubation time of 48 h; or (ii) a seeding density of 1 × 104 parasites/ml and an incubation time of 72 h. Subsequent validation experiments revealed that the 48-h incubation/3 × 104 parasites/ml seeding density condition had a greater sensitivity to detect drug resistance than the 72-h condition. The TSF assay also exhibited high efficiency in identifying parasite drug resistance, as evidenced by its strong correlation with the standard MLC assay results (P = 0.003).ConclusionsThis study presents a robust TSF assay that has the potential to facilitate high-throughput, automated in vitro anti-trichomoniasis susceptibility testing for drug resistance monitoring and drug development. In comparison to the standard MLC method, this assay offers the advantages of reduced labor and elimination of subjective examination.

Dataset Information

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands.

Publications

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands.

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