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 μM concentration, respectively. Conversely, distamycin-A showed an affinity for EcoRI (5’-AAATGC-3’) restriction sites at a concentration of 10 μM. 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.