Development of an automated assay for accelerated in vitro detection of DNA adduct-inducing and crosslinking agents
- Author(s)
- Medan, J; Sleebs, BE; Lackovic, K; Watson, KG; Evison, BJ; Phillips, DR; Cutts, SM;
- Journal Title
- Bioorganic & Medicinal Chemistry Letters
- Abstract
- 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 (32)P-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.
- Publisher
- eLife Sciences
- Keywords
- DNA adducts; DNA-Bind microplates; anthracenediones; anthracyclines; crosslinking agents; europium fluorescence; formaldehyde; intercalation; screening assay
- Research Division(s)
- Chemical Biology
- PubMed ID
- 33486050
- Publisher's Version
- https://doi.org/10.1016/j.bmcl.2021.127813
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2021-03-09 08:05:33
Last Modified: 2021-03-09 08:15:59