A Qualitative and Quantitative Assay to Study DNA/Drug Interaction Based on Sequence Selective Inhibition of Restriction Endonucleases

A Dixit,L Chauhan,R Barthwal,Sa Hassan

doi:10.4314/tjpr.v11i5.4

Abstract

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 bindingspecificity of anticancer drugs.Keywords: Restriction endonucleases, Restriction sites, Anticancer drugs, Restriction inhibition assay (RIA), Binding specificity.

Highlights

Interaction of some anti-tumor agents with DNA has been the subject of intensive research
Considerable amount of effort has been made to elucidate the molecular basis of action of several drugs such as adriamycin, daunomycin, distamycinA, mitoxantrone, berberine and palmatine, which are known to bind to DNA
The results on the detection of binding of Distamycin-A, Adriamycin and Daunomycin to DNA by restriction inhibition assay (RIA).are shown in Fig 1 - 3

Summary

Introduction

Interaction of some anti-tumor agents with DNA has been the subject of intensive research In this regard, considerable amount of effort has been made to elucidate the molecular basis of action of several drugs such as adriamycin, daunomycin, distamycinA, mitoxantrone, berberine and palmatine, which are known to bind to DNA. Considerable amount of effort has been made to elucidate the molecular basis of action of several drugs such as adriamycin, daunomycin, distamycinA, mitoxantrone, berberine and palmatine, which are known to bind to DNA These drugs have good anti-tumor activity and this activity has been correlated to sequence specific binding to DNA. In this context restriction inhibition assay showed more specificity for CA- than GC-rich sequence for adriamycin and more affinity towards GC than AT rich sequence for daunomycin [5], distamycin-A showed more specificity for AT rich sequence [6]

Results

Discussion

Conclusion