A Qualitative and Quantitative Assay to Study DNA/Drug Interaction Based on Sequence Selective Inhibition of Restriction Endonucleases
Purpose: To explore the use of restriction inhibition assay (RIA) to study the binding specificity of some anticancer drugs.
Methods: A 448 bp DNA fragment derived from pBCKS+ plasmid (harboring the polylinker region with multiple restriction endonuclease sites) was used as a template for sequence selective inhibition of the test drugs. The template was incubated with different concentrations of anticancer drugs (adriamycin, daunomycin, mitoxantrone, distamycin-A, berberine and palmatine) prior to digestion with restriction endonucleases - HindIII, EcoRI and EcoRV.
Results: Mitoxantrone, adriamycin and daunomycin showed specificity for HindIII restriction site (5’- AAGCTT-3’) at 220, 100 and 100 ìM concentration, respectively. Conversely, distamycin-A showed an affinity for EcoRI (5’-AAATGC-3’) restriction sites at a concentration of 10 ìM. No binding was observed for berberine and palmatine at a maximum concentration of 2 mM at HindIII, EcoRI and EcoRV restriction sites, respectively.
Conclusion: The inhibition of endonucleases by mitoxantrone, adriamycin, daunomycin, distamycin-A,provides direct evidence of the co-existence of concentration and sequence specificity for drug-DNA interaction as well as the need to explore the possible use of RIA for demonstrating the binding
specificity of anticancer drugs.
Keywords: Restriction endonucleases, Restriction sites, Anticancer drugs, Restriction inhibition assay (RIA), Binding specificity.
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