A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions

Lopez-Crapez, Evelyne; Malinge, Jean-Marc; Gatchitch, François; Casano, Laetitia; Langlois, Thierry; Pugnière, Martine; Roquet, Françoise; Mathis, Gérard; Bazin, Hervé

doi:10.1016/j.ab.2008.09.004

Cited by 14 publications

(10 citation statements)

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“…The detection of mutation based on hybridization alone suffers from high false-positive rates, the use of a protein as auxiliary can improve the specificity, and this was exemplified using mutS, which is a protein involved in DNA mismatch repair processes. An indirect HTRF assay format was designed using 6-His-tagged mutS and XL665-labeled anti-(6-His) mAb in association with 1 –SA conjugate and biotinylated 21-mer DNA duplexes . This assay was used to analyze the interactions between mutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin.…”

Section: Biochemical Applicationsmentioning

confidence: 99%

“…An indirect HTRF assay format was designed using 6-His-tagged mutS and XL665-labeled anti-(6-His) mAb in association with 1−SA conjugate and biotinylated 21-mer DNA duplexes. 53 This assay was used to analyze the interactions between mutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin. A semidirect DNA/mutS HTRF assay using a 1−mutS conjugate was also tested.…”

Section: Chart 1 Europium Macrocycles Studied In This Papermentioning

confidence: 99%

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Luminescent Lanthanide Cryptates: from the Bench to the Bedside

et al. 2014

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The design and application of luminescent lanthanide cryptates for sensing biological interactions is highlighted through the review of the work performed in our laboratory and with academic collaborations. The path from the initial applications probing biochemical interaction in vitro to "state-of-the-art" cellular assays toward clinical applications using homogeneous time-resolved fluorescence technology is described. An overview of the luminescent lanthanide macrocyclic compounds developed at Cisbio in the recent past is given with an emphasis on specific constraints required by specific applications. Recent assays for drug-discovery and diagnostic purposes using both antibody-based and suicide-enzyme-based technology are illustrated. New perspectives in the field of molecular medicine and time-resolved microscopy are discussed.

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Section: Biochemical Applicationsmentioning

confidence: 99%

Section: Chart 1 Europium Macrocycles Studied In This Papermentioning

confidence: 99%

Luminescent Lanthanide Cryptates: from the Bench to the Bedside

et al. 2014

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“…The MutS protein is part of the dam-directed MutHLS mismatch repair pathway in Escherichia coli . By using directly labeled MutS with europium cryptate, Lopez-Crapez et al have developed an HTRF assay to measure interactions between MutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin [66]. …”

Section: Htrf Assay Applicationsmentioning

confidence: 99%

HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications

Degorce

Card²,

Soh³

et al. 2009

TOCHGENJ

399

285

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HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

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“…The low-molecular mass BPLPs can be made to be water-insoluble or -soluble maximizing their potential applications in biological labeling and imaging. The water soluble low-molecular-weight BPLPs may potentially be used for single molecule labeling such as protein and DNA labeling in proteomics and genomics research, where quantum dots may not be ideal because of their size (7,8). The BPLP family may also be suitable for use in fluorescence resonance energy transfer (FRET) (5), 2-photon excited fluorescence microscopy (6), multimodal compositions (combined with magnetic or radionuclear agents) (31), and biosensors (32).…”

Section: Cytotoxicity Evaluation and Bioimaging Study In Vitro And Inmentioning

confidence: 99%

Development of aliphatic biodegradable photoluminescent polymers

Yang

Zhang

Gautam

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

219

271

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Fig. 1. Labeling of the liposome. (A)Outline of the method. Cells were rapidly frozen, freeze-fractured, and evaporated with carbon (C) and platinum/carbon (Pt/C) in vacuum. The replica of the split membrane was digested with SDS to remove noncast molecules and labeled by GST-PH. Both the cytoplasmic and exoplasmic halves of the membrane were examined. (B) Labeling of small unilamellar liposome replicas. Freeze-fracture replicas of liposomes containing 95 mol % of phosphatidylcholine (PC) and 5 mol % of phosphatidylinositol or a phosphoinositide were labeled. Only liposomes containing PI(4,5)P 2 were labeled intensely by GST-PH. A PH mutant, GST-PH (K30N, K32N), which does not bind PI(4,5)P 2, showed little labeling in the PI(4,5)P 2-containing liposome. (C) Quantification of the GST-PH labeling in the liposomes. The number of gold particles per 1 m 2 of the liposome surface is shown (blue). The labeling on the convex (green) and concave (yellow) surfaces showed equivalent results.

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A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions

Cited by 14 publications

References 25 publications

Luminescent Lanthanide Cryptates: from the Bench to the Bedside

Luminescent Lanthanide Cryptates: from the Bench to the Bedside

HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications

Development of aliphatic biodegradable photoluminescent polymers

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