Restriction Inhibition Assay: A Qualitative and Quantitative Method to Screen Sequence Specific DNA Binder from Herbal Plants
Purpose: To employ restriction inhibition assay (RIA) to screen phytochemical-rich fractions (PRFs) with high affinity for EcoRI and HindIII restriction sequences and correlate their interaction to an anticancer activity.
Methods: pBR322 linear plasmid DNA was used as a template to screen the sequence-selective inhibition of aqueous extracts of Cinnamomum zeylanicum and Picrorhiza kurroa, respectively. The template was further incubated with different concentrations of PRFs prior to digestion with restriction endonucleases HindIII and EcoRI. The Expressed Sequence Tags (ESTs) and Sequence Tag Sites (STS) of oncogenes were screened for the presence of EcoRI and HindIII restriction sequences to associate an anticancer property to PRF.
Results: The inhibitory concentrations of Cinnamomum zeylanicum aqueous extract against HindIII and EcoRI endonucleases were approximately 2.5 and 5 μg/μl, respectively. No binding was observed for Picrorhiza kurroa at both HindIII and EcoRI restriction sites. The saponin-rich fractions of Cinnamomum zeylanicum showed significant (p < 0.001) inhibition as compared to control at concentrations of 0.28±1.45 μg/μl for EcoRI and 0.11±2.68 μg/μl for HindIII endonucleases. Both EcoRI and HindIII restriction sites were found repeatedly in the STS and ESTs of BRCA2, the early onset oncogene.
Conclusion: The inhibition of endonucleases by phytochemical-rich fractions provides direct evidence of the use of RIA for screening as well as demonstrating the binding specificity of these PRFs. The presence of 5’-AAGCTT-3’ & 5’-GAATTC-3’ in the ESTs of BRAC2 provides an insight into the use of screened components as leads in the search for novel anticancer compounds.
Keywords: Restriction endonucleases, Restriction sites, Phytochemicals, Restriction inhibition assay (RIA), Binding specificity, Oncogenes, Sequence tag sites, Expressed sequence tag, Anticancer.