A high-throughput resonance energy transfer assay for Staphylococcus aureus DNA ligase

Benson, Ericka L; Tomich, Paul K.; Wolfe, Mark L.; Choi, Gil H.; Hagadorn, Jeanne C.; Mutchler, Veronica T.; Garlick, Robert

doi:10.1016/j.ab.2003.09.019

Cited by 11 publications

(11 citation statements)

References 8 publications

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“…Radioactive assays need special facilities and personnel training; furthermore, radiolabeled g-[P 32 ] has a short half-life and besides its health hazard-related issue, it also requires expensive waste disposal of radioactivity. Fluorescence resonance energy transfer (FRET) and time-resolved FRET-based assays 16,37 for DNA ligases have been designed and require labeled DNA oligos and significant optimization of assay conditions. Bioluminescent assays allow a large dynamic range, with high signal:background ratio and S/N ratio, making the assay highly sensitive, requiring low amounts of enzymes, and with no additional requirement for labeling, making these assays simple, homogenous, and amenable to high-throughput screening.…”

Section: Resultsmentioning

confidence: 99%

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions

Mondal

Hsiao

Goueli

2017

ASSAY and Drug Development Technologies

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Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD and enzymes that use NAD like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

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Section: Resultsmentioning

confidence: 99%

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions

Mondal

Hsiao

Goueli

2017

ASSAY and Drug Development Technologies

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“…The traditional methods for studying hairpin DNAs are based on fluorescent and electrochemical measurements. In these studies, it was found that there is a conformation change of hairpin DNA during the detection process (Benson et al, 2004;Chen et al, 2002;Liu et al, 2005;Vacek et al, 2008;Zauner et al, 2005). By taking advantage of this conformation change, we firstly realized the hairpin DNAs study using SPR and found that the conformation change of hairpin DNAs can result in a large change in SPR dip shift (Luan et al, 2010a,b).…”

Section: Introductionmentioning

confidence: 96%

Au-NPs enhanced SPR biosensor based on hairpin DNA without the effect of nonspecific adsorption

Luan

Zhou

Tan

et al. 2011

Biosensors and Bioelectronics

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“…The method was similar to that previously described (20,21 The mixture was reacted at room temperature for 20 min, then 30 µl Quench reagent (8 M urea, 1 M Tris and 20 mM EDTA in water) was added as the reaction terminating agent. Plates were read using a Tecan Ultra plate reader (Tecan Group Ltd., Männedorf, Switzerland), with monitoring of the ratio of fluorescence intensities at two emission wavelengths (the TAMRA acceptor at 595 nm and the FAM donor at 535 nm) upon excitation of the FAM donor at 485 nm.…”

Section: Methodsmentioning

confidence: 99%

Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+-dependent DNA ligase inhibitor

Zhou

Cai

et al. 2016

Experimental and Therapeutic Medicine

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Abstract. Cordycepin exhibits various bio-activities, including anticancer, antibacterial, antiviral and immune regulation activities, and is a significant focus of research. However, the preparation of high-purity cordycepin remains challenging. Also, the molecular target with which cordycepin interacts to cause an antibacterial effect remains unknown. In the present study, cordycepin was prepared by preparative high-performance liquid chromatography (prep-HPLC) and the purity obtained was 99.6%, indicating that this technique may be useful for the large-scale isolation of cordycepin in the future. The results of computational molecular docking analysis indicated that the interaction energy between cordycepin and NAD + -dependent DNA ligase (LigA) was lower than that between cordycepin and other common antibacterial targets. The highly pure cordycepin obtained by prep-HPLC demonstrated inhibitory activity against LigA from various bacteria in vitro. In conclusion, cordycepin may be useful as a broad-spectrum antibiotic targeting LigA in various bacteria. IntroductionCordycepin is an antimicrobial substance that was first isolated from the entomopathogenic fungus Cordyceps militaris by Cunningham et al in 1950 (1). Since its discovery, a number of studies have focused on this substance (2,3). Cordycepin ( Fig. 1A) with the molecular formula C 10 H 13 N 5 O 3 has been demonstrated to have various bio-activities, including anticancer (4-7), antibacterial (8), antiviral (9,10) and immune regulation activities (11) and has became a topic of interest to various research groups.However, highly pure cordycepin is challenging to obtain; various drawbacks have been identified in the purification techniques of ion resin adsorption (12,13), silica gel column chromatography (14) and supercritical extraction (15). The high price of cordycepin acts as a significant barrier to the study of its bio-activity. Preparative high performance liquid chromatography (prep-HPLC) can shorten the production cycle, has relatively low operating costs, and is simple to carry out. Therefore, it is commonly used for the purification of chemical drugs and biological drugs by pharmaceutical companies and particularly in industrial purification (16). Prep-HPLC can be used to provide purified samples on a kilogram, or even ton, scale. Also, prep-HPLC is an effective technology currently used for natural product isolation in industry (17). The present study describes a new method of obtaining high-purity cordycepin by prep-HPLC, which may be useful for the large-scale isolation of cordycepin in the future.Although studies have shown that cordycepin is a broad-spectrum antibiotic (18), studies focusing on the mechanism of the antibacterial activity and the possible targets are lacking. Molecular modeling and docking technology, which is efficient, accurate and simple to use, can reproduce the true interaction process between drug and targets that some of the current laboratory techniques are unable to detect, and is a method for predicting the mecha...

show abstract

A high-throughput resonance energy transfer assay for Staphylococcus aureus DNA ligase

Cited by 11 publications

References 8 publications

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions

Au-NPs enhanced SPR biosensor based on hairpin DNA without the effect of nonspecific adsorption

Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+-dependent DNA ligase inhibitor

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