Restriction Inhibition Assay: A Qualitative and Quantitative Method to Screen Sequence Specific DNA Binder from Herbal Plants

摘要

Purpose: To employ restriction inhibition assay (RIA) to screen phytochemical-rich fractions (PRFs) with high affinity for Eco R I and Hin d III 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 Hin d III and Eco R I. The Expressed Sequence Tags (ESTs) and Sequence Tag Sites (STS) of oncogenes were screened for the presence of Eco R I and Hin d III restriction sequences to associate an anticancer property to PRF. Results: The inhibitory concentrations of Cinnamomum zeylanicum aqueous extract against Hin d III and Eco R I endonucleases were approximately 2.5 and 5 μg/μl, respectively. No binding was observed for Picrorhiza kurroa at both Hin d III and Eco R I restriction sites. The saponin-rich fractions of Cinnamomum zeylanicum showed significant (p R I and 0.11±2.68 μg/μl for Hin d III endonucleases. Both Eco R I and Hin d III 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.