Immobilized DNA hairpins for assay of sequential breaking and joining of DNA backbones

Scott, Benjamin O.S.; Lavesa-Curto, Manuel; Bullard, Desmond R.; Butt, Julea N.; Bowater, Richard P.

doi:10.1016/j.ab.2006.08.010

Cited by 7 publications

(9 citation statements)

References 32 publications

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“…A brief flash of chemiluminescence occurred when the remaining acridinium ester was exposed to h 2 o 2 and Naoh. scott et al 15 immobilized biotinylated DNA hairpins on streptavidin-coated assay plates. A fluorescently labeled oligonucleotide was annealed to the single-stranded part of the hairpin to form a nick.…”

Section: Resultsmentioning

confidence: 99%

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A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase

et al. 2011

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DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5′-PO4 and 3′-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD+-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer–based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD+-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

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Section: Resultsmentioning

confidence: 99%

“…moreover, detection of AtP by luciferase is prone to a high false-positive rate in high-throughput screening. 16 the use of a streptavidincoated plate to capture biotinylated, ligated DNA 8,15 requires expensive assay plates and includes a washing step. the chemiluminescence assay of gul et al…”

Section: Resultsmentioning

confidence: 99%

A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase

et al. 2011

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“…Gel electrophoresis is a convenient method for detecting the sizes of molecules, and it has been widely used among the primary biochemical studies of DNA ligases that are reviewed here (e.g. see [ 64 , 66 , 67 ]). However, gel electrophoresis is rather time-consuming and laborious and, thus, is not ideal for drug discovery purposes.…”

Section: Assays Of Dna Joining By Dna Ligasesmentioning

confidence: 99%

“…A wide range of biophysical chemistry approaches have been used to detect the joining of DNA strand breaks. Those applied to NDLs or ADLs from bacteria or archaea include use of fluorescently-labelled probes [ 66 , 70 , 72 , 73 ], FRET [ 69 , 74 – 77 ], fluorescence quenching and molecular beacon-based approaches [ 78 – 81 ], electrochemical methods [ 67 , 79 , 81 – 84 ], a nanoparticle-based sensor [ 85 ] and surface plasmon resonance [ 86 ]. Biochemical analyses of the ligation reaction are facilitated by assays that do not require ‘labelling’ of the nucleic acid, such as label-free electrochemical approaches using mercury-based electrodes and nicked plasmid DNA substrates [ 67 ].…”

Section: Assays Of Dna Joining By Dna Ligasesmentioning

confidence: 99%

Biochemical and structural characterization of DNA ligases from bacteria and archaea

2016

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“…The methods now used to assay ligase activity—which rely on gel electrophoresis of labeled oligonucleotides—have a limited throughput and are tedious when applied for profiling the activity of the ligase for a family of substrates (16). Efforts to increase throughput have adopted homogeneous phase assays that use a secondary reaction to detect the product of the ligase reaction (17), but the use of labels and multiple steps increase the incidence of false positive and negative results.…”

Section: Introductionmentioning

confidence: 99%

Profiling the selectivity of DNA ligases in an array format with mass spectrometry

Kim¹,

Mrksich²

2009

Nucleic Acids Research

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This article describes a method for the global profiling of the substrate specificities of DNA ligases and illustrates examples using the Taq and T4 DNA ligases. The method combines oligonucleotide arrays, which offer the benefits of high throughput and multiplexed assays, with mass spectrometry to permit label-free assays of ligase activity. Arrays were prepared by immobilizing ternary biotin-tagged DNA substrates to a self-assembled monolayer presenting a layer of streptavidin protein. The array represented complexes having all possible matched and mismatched base pairs at the 3′ side of the nick site and also included a number of deletions and insertions at this site. The arrays were treated with ligases and adenosine triphosphate or analogs of the nucleotide triphosphate and then analyzed by matrix-assisted laser desorption-ionization mass spectrometry to determine the yields for both adenylation of the 5′-probe strand and joining of the two probe strands. The resulting activity profiles reveal the basis for specificity of the ligases and also point to strategies that use ATP analogs to improve specificity. This work introduces a method that can be applied to profile a broad range of enzymes that operate on nucleic acid substrates.

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Immobilized DNA hairpins for assay of sequential breaking and joining of DNA backbones

Cited by 7 publications

References 32 publications

A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase

A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase

Biochemical and structural characterization of DNA ligases from bacteria and archaea

Profiling the selectivity of DNA ligases in an array format with mass spectrometry

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