Development of an automated assay for accelerated in vitro detection of DNA adduct-inducing and crosslinking agents
| Autor: | Keith G. Watson, Benny J. Evison, Brad E. Sleebs, Don R. Phillips, Jelena Medan, Suzanne M. Cutts, Kurt Lackovic |
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| Rok vydání: | 2020 |
| Předmět: |
Clinical Biochemistry
Intercalation (chemistry) Pharmaceutical Science 01 natural sciences Biochemistry Adduct chemistry.chemical_compound Automation DNA Adducts Structure-Activity Relationship Drug Development Drug Discovery DNA adduct DNA Interstrand Crosslinking Denaturation (biochemistry) Molecular Biology Chromatography Dose-Response Relationship Drug Molecular Structure 010405 organic chemistry Oligonucleotide Organic Chemistry Fluorescence 0104 chemical sciences 010404 medicinal & biomolecular chemistry Cross-Linking Reagents chemistry Molecular Medicine DNA |
| Zdroj: | Bioorganicmedicinal chemistry letters. 35 |
| ISSN: | 1464-3405 |
| Popis: | Current techniques for the identification of DNA adduct-inducing and DNA interstrand crosslinking agents include electrophoretic crosslinking assays, electrophoretic gel shift assays, DNA and RNA stop assays, mass spectrometry-based methods and 32P-post-labelling. While these assays provide considerable insight into the site and stability of the interaction, they are relatively expensive, time-consuming and sometimes rely on the use of radioactively-labelled components, and thus are ill-suited to screening large numbers of compounds. A novel medium throughput assay was developed to overcome these limitations and was based on the attachment of a biotin-tagged double stranded (ds) oligonucleotide to Corning DNA-Bind plates. We aimed to detect anthracycline and anthracenedione DNA adducts which form by initial non-covalent intercalation with duplex DNA, and subsequent covalent adduct formation which is mediated by formaldehyde. Following drug treatment, DNA samples were subjected to a denaturation step, washing and then measurement by fluorescence to detect remaining drug-DNA species using streptavidin-europium. This dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) is a time-resolved fluorescence intensity assay where the fluorescence signal arises only from stabilised drug-DNA complexes. We applied this new methodology to the identification of anthracycline-like compounds with the ability to functionally crosslink double-strand oligonucleotides. The entire procedure can be performed by robotics, requiring low volumes of compounds and reagents, thereby reducing costs and enabling multiple compounds to be assessed on a single microtitre plate. |
| Databáze: | OpenAIRE |
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